HPV vaccines and methods of use thereof

ABSTRACT

Methods for generating immune responses using adenovirus vectors that allow multiple vaccinations with the same adenovirus vector and vaccinations in individuals with preexisting immunity to adenovirus are provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/622,263, filed on Sep. 18, 2012, which is a continuation of U.S.application Ser. No. 12/651,836, filed Jan. 4, 2010, now issued as U.S.Pat. No. 8,298,549 on Oct. 30, 2012, which is a continuation-in-part ofInternational Application No. PCT/US2008/068924, filed on Jul. 1, 2008,which claims the benefit of priority to U.S. Provisional Application No.60/947,601, filed on Jul. 2, 2007, all of which are incorporated hereinby reference in their entireties.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Contract No. 1 R43AI071733-01 awarded by the National Institutes of Health, NationalInstitutes of Allergy and Infectious Diseases; Contract No. 2R44AI1071733-03 awarded by the National Institutes of Health; Contract No.1R43CA 134063-01 awarded by the National Institutes of Health; andContract No. 1R43CA 139663-01 awarded by the National Institutes ofHealth. The government may have certain rights in this invention.

BACKGROUND

Technical Field

The present invention relates to methods for generating immune responsesusing adenovirus vectors that allow for multiple vaccination regimens.

Description of the Related Art

The most difficult problem with adenovirus vectors has been theirinability to sustain long-term transgene expression due largely to thehost immune response that eliminates the adenovirus vector and virallytransduced cells in immune-competent subjects. Thus, the use of FirstGeneration adenovirus vector vaccines is severely limited by preexistingor induced immunity of vaccines to adenovirus (Ad) (Yang, et al. J Virol77/799-803 (2003); Casimiro, et al. J Virol 77/6305-6313 (2003)). Onegroup reported that a preponderance of humans have antibody againstadenovirus type 5 (Ad5), the most widely used serotype for gene transfervectors, and that two-thirds of humans studied have lympho-proliferativeresponses against Ad (Chirmule, et al. Gene Ther 6/1574-1583 (1999)). Inanother study, an adenovirus vector vaccine carrying an HIV-1 envelopegene was incapable of reimmunizing a primed immune response usingnon-adjuvanted DNA (Barouch, et al. J. Virol 77/8729-8735 (2003)).Another group reported that non-human primates having pre-existingimmunity against Ad5 due to a single immunization with Ad5 were unableto generate transgene-specific antibodies to HIV proteins, as well asaltering the overall T cell responses (McCoy, et al. J Virol81/6594-6604 (2007)).

There are numerous mechanisms by which preexisting immunity interfereswith adenovirus vector vaccines but the simplest is the presence ofneutralizing antibody followed by cell mediated immune elimination of Adinfected antigen harboring cells. Both of these responses are directedto several Ad proteins. Several approaches have been proposed toovercome the barrier of preexisting anti-vector immunity. Perhaps themost straightforward approach would be to increase the vector vaccinedose. Although there is evidence that increasing vaccine doses canincrease induction of desired cell mediated immune (CMI) responses inAd-immune animals (Barouch, et al. J. Virol 77/8729-8735 (2003)), itoften results in unacceptable adverse effects in animals and humans.Consequently, most investigators using First Generation Ad5 vectorvaccines use the approach of a heterologous prime-boost regimen, usingnaked (non-vectored) DNA as the priming vaccination, followed by an Ad5vector immunization. This protocol also results in a subsequent immuneresponse against Ad5 such that one cannot administer a furtherre-immunization (boost) with the same (or a different) adenovirus vectorvaccine that utilizes the same viral backbone. Therefore, with thecurrent First Generation of Ad5 vectors, using this approach alsoabrogates any further use of Ad5 vector immunization in the Ad5immunized vaccinee.

First Generation (E1 deleted) adenovirus vector vaccines express Ad lategenes, albeit at a decreased level and over a longer time period thanwild-type Ad virus (Nevins, et al. Cell 26/213-220 (1981); Gaynor, etal. Cell 33/683-693 (1983); Yang, et al. J Virol 70/7209-7212 (1996)).When using First Generation adenovirus vectors for immunization, vaccineantigens are presented to the immune system simultaneously with highlyimmunogenic Ad capsid proteins. The major problem with these adenovirusvectors is that the immune responses generated are less likely to bedirected to the desired vaccine epitopes (McMichael, et al. Nat RevImmunol 2/283-291 (2002)) and more likely to be directed to theadenovirus-derived antigens, i.e., antigenic competition. There iscontroversy about the mechanism by which First Generation adenovirusvectors are potent immunogens. It has been hypothesized that thecomposition of the Ad capsid or a toxic effect of viral genes createsgeneralized inflammation resulting in a nonspecific immune stimulatoryeffect. The E1 proteins of Ad act to inhibit inflammation followinginfection (Schaack, et al. PNAS 101/3124-3129 (2004)). Removal of thegene segments for these proteins, which is the case for First Generationadenovirus vectors, results in increased levels of inflammation(Schaack, et al. PNAS 101/3124-3129 (2004); Schaack, et al. ViralImmunol 18/79-88 (2005)). It has been reported that adenovirus vectorsefficiently infect antigen-presenting cells (APC) such as dendriticcells and less immunogenic viral vectors do not (Jooss, et al. J Virol72/4212-4223 (1998)). Antigen presenting cells (APC), such as dendriticcells, are responsible for initiation of CMI responses (Kirk, et al. HumGene Ther 11/797-806 (2000)). It has been reported that prevention ofgene expression in dendritic cells greatly reduces the intensity of theCMI response (Hartigan-O'Connor, et al. Mol Ther 4/525-533 (2001)).

Thus, it is apparent that there remains a need for a more effectivevaccine vector candidate. In particular, there remains a need in the artfor Ad vaccine vectors that allow multiple vaccinations and vaccinationsin individuals with preexisting immunity to Ad. In addition, there is nohomologous vaccine delivery vector that can be employed in a primereimmunization protocol for vaccination. The present invention providesthis and other advantages.

BRIEF SUMMARY

One aspect of the invention provides a method of generating an immuneresponse against one or more target antigens in an individual comprisingadministering to the individual an adenovirus vector comprising: a) areplication defective adenovirus vector, wherein the adenovirus vectorhas a deletion in the E2b region, and b) a nucleic acid encoding the oneor more target antigens; and readministering the adenovirus vector atleast once to the individual; thereby generating an immune responseagainst the one or more target antigens.

Another aspect of the invention provides a method for generating animmune response against one or more target antigens in an individual,wherein the individual has preexisting immunity to adenovirus,comprising: administering to the individual an adenovirus vectorcomprising: a) a replication defective adenovirus vector, wherein theadenovirus vector has a deletion in the E2b region, and b) a nucleicacid encoding the one or more target antigens; and readministering theadenovirus vector at least once to the individual; thereby generating animmune response against the one or more target antigens.

In one embodiment of the methods described herein, the target antigencomprises an HIV protein, a human papilloma virus protein, a herpessimplex virus protein, a hepatitis C virus protein, a malaria protein, aplague protein, a M. tuberculosis protein, or a Streptococcus pneumoniaprotein, or an immunogenic fragment or variant thereof. In certainembodiments, the HIV protein is an HIV-gag protein. In a furtherembodiment, the target antigen comprises an antigen derived from aVenezuelan Equine Encephalitis Virus (VEEV), Western Equine EncephalitisVirus, or Japanese Encephalitis Virus protein. In yet furtherembodiments, the target antigen comprises a Leishmania protein, a cancerprotein such as carcinoembryonic antigen, Her2/Neu, a human papillomavirus protein, or WT-1, or a variant or fragment thereof. In aparticular embodiment, the human papilloma virus protein is E6. Inanother embodiment, the human papilloma virus protein is E7. In certainembodiments, the target antigen is a variant having one or more reducedbiological activities as compared to the wild type target antigen. Inparticular embodiments, a target antigen is a variant that is modifiedto have reduced oncogenicity as compared to the wild type targetantigen.

In one embodiment, the target antigen comprises an antigen derived froman influenza virus protein, or a variant or fragment thereof. In thisregard, the influenza protein may be derived from the H5N1 influenzavirus. In a further embodiment, the influenza virus protein may bederived from any influenza virus, including but not limited to H3N2,H9N1, H1N1, H2N2, H7N7, H1N2, H9N2, H7N2, H7N3, or H10N7. In certainembodiments, the influenza virus protein may be any influenza protein,including but not limited to, haemagglutinin, neuraminidase, or matrixprotein M1.

A further aspect of the invention provides a method of generating animmune response against one or more target antigens in an individualcomprising: administering to the individual a first adenovirus vectorcomprising: a) a replication defective adenovirus vector, wherein theadenovirus vector has a deletion in the E2b region, and b) a nucleicacid encoding at least one target antigen; and administering to theindividual a second adenovirus vector comprising: a) a replicationdefective adenovirus vector, wherein the adenovirus vector has adeletion in the E2b region, and b) a nucleic acid encoding at least onetarget antigen, wherein the at least one target antigen of the secondadenovirus vector is the same or different from the at least one targetantigen of the first adenovirus vector; thereby generating an immuneresponse against one or more target antigens.

In one embodiment of the methods provided herein, the adenovirus vectoris not a gutted vector. In another embodiment of the methods providedherein, the individual has preexisting immunity to adenovirus. In afurther embodiment, the at least one target antigen of the first and thesecond adenovirus vectors are derived from the same infectious organism.In another embodiment, the at least one target antigen of the first andthe second adenovirus vectors are derived from different infectiousorganisms.

In yet another embodiment of the methods of the invention, the at leastone target antigen of the first adenovirus vector comprises an HIVprotein. In a further embodiment, the at least one target antigen of thefirst adenovirus vector comprises an HIV protein and the at least onetarget antigen of the second adenovirus vector comprises an HIV protein.In another embodiment, the at least one target antigen of the firstadenovirus vector comprises an HIV protein and the at least one targetantigen of the second adenovirus vector comprises an HIV protein that isdifferent from the HIV protein of the first adenovirus vector. In afurther embodiment, the HIV protein of the first or second adenovirusvector is an HIV-gag protein. In a yet further embodiment, the firstadenovirus vector comprises an HIV-gag protein and the second adenovirusvector comprises β-galactosidase. In certain embodiments, theβ-galactosidase is an E. coli β-galactosidase.

In certain embodiments of the methods provided herein, the at least onetarget antigen of the first adenovirus vector comprises a cancer protein(e.g., a Her2/neu antigen, a human papilloma virus protein or acarcinoembryonic protein), or a fragment or variant thereof. In otherembodiments, the at least one target antigen of the first adenovirusvector comprises a bacterial antigen, a viral antigen, an antigenderived from a protozoan protein, an antigen derived from a fungalprotein, an antigen derived from a mold protein, an antigen derived fromany mammalian protein, or an antigen derived from an avian protein, or afragment or variant thereof. In certain embodiments, the target antigenis a variant having one or more reduced biological activities ascompared to the wild type target antigen. In particular embodiments, atarget antigen is a variant that is modified to have reducedoncogenicity as compared to the wild type target antigen.

In a further related embodiment, the present invention includes anadenovirus vector comprising: i) a replication defective adenovirusvector, wherein the adenovirus vector has a deletion in the E2b region,and a nucleic acid encoding one or more target antigens, wherein saidone or more target antigens comprises a modified cancer protein havingone or more reduced activities. In one embodiment, the modified cancerprotein has reduced oncogenic activity. In one embodiment, the modifiedcancer protein is a modified Her2/neu protein having reduced kinaseactivity. In one embodiment, the modified cancer protein is a modifiedhuman papillomavirus E6 protein having reduced binding to p53. In oneembodiment, the modified cancer protein is a modified humanpapillomavirus E7 protein having reduced binding to Rb.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a bar graph showing antibody levels from mice immunized withAd5Null. Mice were immunized three times with Ad5Null viral particles at14 day intervals. Note the presence of increasing anti-Ad antibodylevels after each immunization.

FIG. 2 is a bar graph showing neutralizing antibody levels from miceimmunized with Ad5Null. Mice were immunized three times with Ad5Nullviral particles at 14 day intervals. Note the presence of increasingneutralizing antibodies after each immunization. Optical densityreadings indicate the presence of viable target cells.

FIG. 3 shows the measured levels of antibody in immunized mice. Micewere injected three times with E2b deleted adenovirus vector containingthe HIV-gag gene. Note the presence of significantly (P<0.05) elevatedlevels of Gag IgG antibody in experimental mice as compared to normalcontrol mice. Horizontal bars represent the mean value.

FIG. 4 is a graph showing antibody levels over time in mice immunizedwith two different E2b deleted adenovirus vectors each with a differenttarget antigen. Mice were immunized with E2b deleted adenovirus vectorcontaining the HIV-gag gene three times at 14 day intervals. Four weekslater, the same group of mice was immunized two additional times at 14day intervals with E2b deleted adenovirus vector containing theEscherichia coli β-galactosidase gene. Note the presence of increasinglevels of HIV-Gag IgG antibody levels after multiple immunizations ascompared to pre-immunization levels. Moreover, note the presence ofincreasing levels of β-galactosidase IgG antibody after two injectionsin the same group of mice as compared to pre-immunization levels.

FIG. 5A and FIG. 5B are bar graphs showing numbers of T-cells expressingIFN-γ and IL-2, respectively. Mice were immunized with E2b deletedadenovirus vector containing the HIV-gag gene three times at 14 dayintervals. Four weeks later, the same group of mice was immunized twoadditional times at 14 day intervals with E2b deleted adenovirus vectorcontaining the β-galactosidase gene. To assess cell-mediated immuneresponses, ELISPOT assays were performed to determine the number ofinterferon-v (IFN-γ) or interleukin-2 (IL-2) secreting cells followingstimulation with the HIV-Gag protein, β-galactosidase, or Ad5Null virus.The data are expressed as the number of spot forming cells (SFC) per 10⁶splenocytes. Note the number of IFN-γ and IL-2 producing cells aftersplenocytes were stimulated.

FIG. 6 is a bar graph showing the Ad5 neutralizing antibody (NAb) titersin non-human primates (NHP) during the vaccination protocol using Ad5[E1-, E2b-]-gag. Three NHP were injected with a single dose of 10¹⁰ VPviable wild type Ad5. Ad5 NAb was measured 30 days after administrationand the NHP titers were ≥1:50. The Ad5 immune NHP were then immunizedthree times on days 0, 27, and 58 with Ad5 [E1-, E2b-]-gag (10¹⁰VP/dose). Note the increasing levels of NAb induced during vaccinationwith Ad5 [E1-, E2b-]-gag. Vertical bars indicate the Standard Error ofthe Mean (SEM).

FIG. 7A and FIG. 7B are bar graphs showing numbers of peripheral bloodmononuclear cells (PBMCs) in Ad5 immune cynomolgus macaques secretingIFN-γ and IL-2, respectively. PBMCs from individual NHP were collectedand cell-mediated immune (CMI) responses were assayed 32 days (Day 90)after the final immunization with Ad5 [E1-, E2b-]-gag. Note thesignificantly (P<0.05) elevated levels of IFN-γ (FIG. 7A) and IL-2 (FIG.7B) secreting cells from the PBMC sample taken after the vaccinationprotocol as compared to a baseline sample (Day −8) taken beforevaccinations. The ELISpot data are expressed as the number of spotforming cells (SFC) per 10⁶ PBMCs. Vertical bars indicate the SEM.

FIG. 8 is a Western blot image showing HER2 expression in human cells.Human A-549 cells were infected with saline (A); Ad5 [E1-, E2b-]-HER2 ata multiplicity of infection (MOI) of 900 (B); or Ad5 [E1-, E2b-]-HER2 atan MOI of 150 (C). After 24 hours, total cell protein was harvested andHER2 protein expression was assessed by Western blot.

FIG. 9 is a bar graph showing neutralizing antibody (NAb) titers inBALB/c mice immunized 1, 2, or 3 times with 10¹⁰ Ad5-null VP. Valuesrepresent mean±SEM.

FIG. 10 is a bar graph showing serum Ad5 NAb levels in BALB/c micevaccinated 3 times on days 0, 7, & 17 with 10¹⁰ Ad5 [E1-, E2b-]-HER2.Mice were made Ad5 immune by 2 injections with Ad5-null on days −28 and-14. Note the high levels in NAb activity on day 32 at which timesplenocytes were assessed for CMI responses. Values represent theinverse of the endpoint NAb.

FIG. 11 is a bar graph showing INF-γ and IL-2 secreting splenocytes(SFC) from Ad5 immune mice immunized 1, 2, or 3 times with Ad5 [E1-,E2b-]-HER2. Note the highly elevated CMI responses after the thirdimmunization (3×). For positive controls, splenocytes were exposed toConcanavalin A (Con A) (data not shown). Values represent the mean±SEM.

FIG. 12 is a bar graph showing anti-HER2 IgG antibody levels in Ad5immune mice immunized one (1×), two (2×), or three (3×) times with Ad5[E1-, E2b-]-HER2. Note the increasing antibody levels associated withincreasing immunizations. Serum samples diluted 1/100 and antibodylevels were assessed by ELISA with reference to a standard IgG curve toquantitate IgG antibody levels. Values represent Mean±SEM.

FIG. 13A is a bar graph showing serum AST levels in control mice andmice vaccinated 3 times [HER2 (3×)] with 10¹⁰ viral particles of Ad5[E1-, E2b-]-HER2. Values represent mean±SEM.

FIG. 13B is a bar graph showing serum ALT levels in control mice andmice vaccinated 3 times [HER2 (3×)] with 10¹⁰ viral particles of Ad5[E1-, E2b-]-CEA. Values represent mean±SEM.

FIG. 14 is a line graph showing tumor volume. Ad5 immune mice wereinjected 3 times with Buffer solution, Ad5-Null (empty), or Ad5 [E1,E2b-]-HER2. Two weeks after the last immunization, they were challengedwith HER2 expressing tumors. Note the significant lack of tumor growthin the Ad5 [E1-, E2b-]-HER2 immunized mice. Values represent themean±SEM.

FIG. 15 is a bar graph showing the induction of NAb in C57BL/6 miceafter injections with Ad5-Null vector platform (VP). Note the increasinglevels of NAb induced in mice after repeated injections with Adparticles. Values represent mean±SEM.

FIG. 16A is a bar graph showing INF-γ secreting splenocytes from Ad5immune mice immunized with Ad5 [E1-]-CEA or Ad5 [E1, E2b-]-CEA. Note thesignificantly elevated response in splenocytes from the Ad5 [E1-,E2b]-CEA immunized group. Values represent mean±SEM.

FIG. 16B is a bar graph showing IL-2 secreting splenocytes from Ad5immune mice immunized with Ad5 [E1-]-CEA or Ad5 [E1-, E2b-]-CEA. Notethe significantly elevated response in splenocytes from the Ad5 [E1-,E2b]-CEA immunized group. Values represent mean±SEM.

FIG. 17 is a bar graph showing serum AST levels in control mice and micevaccinated with 10¹⁰ viral particles of Ad5 [E1-]-CEA or Ad5 [E1-,E2b-]-CEA. Values represent mean±SEM.

FIG. 18 is a line graph showing tumor volume. Ad5 immune C57BL/6 micewere injected with MC38 CEA expressing tumor cells and subsequentlytreated (Vac) with Ad5 [E1-, E2b-]-CEA vaccine as described. Note thesignificant reduction in tumor size by days 19-21 as compared tountreated control tumor bearing mice. Tumor measurements were taken andvolumes were determined. Statistical analysis was performed using theBonferroni post-tests analysis with PRISM software. Values representmean±SEM.

FIG. 19 is a graph showing tumor weights from treated and untreated Ad5immune MC38 tumor bearing mice. Note the significant (p=0.0124)reduction in tumor weights from the mice treated with Ad5 [E1-,E2b-]-CEA. Values represent mean±SEM.

FIG. 20 is a bar graph showing INF-γ secreting splenocytes fromnon-human primates (NHP). Groups of NHP were immunized twice at 2 weekintervals with Ad5 [E1-, E2b-]-SIV-gag. After immunizations, PBMCs wereassessed for IFN-γ secreting lymphocytes (SFC). Significantly elevatednumbers of IFN-γ secreting lymphocytes were observed after immunizationsand these levels were similar in the Ad5 immune and naïve groups. Valuesrepresent the mean±SEM.

DETAILED DESCRIPTION

The present invention relates to methods and adenovirus vectors forgenerating immune responses against target antigens. In particular, thepresent invention provides an improved adenovirus (Ad)-based vaccinesuch that multiple vaccinations against one or more antigenic targetentity can be achieved. Importantly, vaccination can be performed in thepresence of preexisting immunity to the Ad and/or administered tosubjects previously immunized multiple times with the adenovirus vectorof the present invention or other adenovirus vectors. The adenovirusvectors of the invention can be administered to subjects multiple timesto induce an immune response against an antigen of interest, includingbut not limited to, the production of antibodies and cell-mediatedimmune responses against one or more target antigens.

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

The term “adenovirus” or “Ad” refers to a group of non-enveloped DNAviruses from the family Adenoviridae. In addition to human hosts, theseviruses can be found in, but are not limited to, avian, bovine, porcineand canine species. The present invention contemplates the use of anyadenovirus from any of the four genera of the family Adenoviridae (e.g.,Aviadenovirus, Mastadenovirus, Atadenovirus and Siadenovirus) as thebasis of an E2b deleted virus vector, or vector containing otherdeletions as described herein. In addition, several serotypes are foundin each species. Ad also pertains to genetic derivatives of any of theseviral serotypes, including but not limited to, genetic mutation,deletion or transposition of homologous or heterologous DNA sequences.

A “helper adenovirus” or “helper virus” refers to an Ad that can supplyviral functions that a particular host cell cannot (the host may provideAd gene products such as E1 proteins). This virus is used to supply, intrans, functions (e.g., proteins) that are lacking in a second virus, orhelper dependent virus (e.g., a gutted or gutless virus, or a virusdeleted for a particular region such as E2b or other region as describedherein); the first replication-incompetent virus is said to “help” thesecond, helper dependent virus thereby permitting the production of thesecond viral genome in a cell.

The term “Adenovirus5 null (Ad5null)”, as used herein, refers to anon-replicating Ad that does not contain any heterologous nucleic acidsequences for expression.

The term “First Generation adenovirus”, as used herein, refers to an Adthat has the early region 1 (E1) deleted. In additional cases, thenonessential early region 3 (E3) may also be deleted.

The term “gutted” or “gutless”, as used herein, refers to an adenovirusvector that has been deleted of all viral coding regions.

The term “transfection” as used herein refers to the introduction offoreign nucleic acid into eukaryotic cells. Transfection may beaccomplished by a variety of means known to the art including calciumphosphate-DNA co-precipitation, DEAE-dextran-mediated transfection,polybrene-mediated transfection, electroporation, microinjection,liposome fusion, lipofection, protoplast fusion, retroviral infection,and biolistics.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign nucleic acid, DNA or RNA, intothe genome of the transfected cell. The term “stable transfectant”refers to a cell which has stably integrated foreign DNA into thegenomic DNA.

The term “reporter gene” indicates a nucleotide sequence that encodes areporter molecule (including an enzyme). A “reporter molecule” isdetectable in any of a variety of detection systems, including, but notlimited to enzyme-based detection assays (e.g., ELISA, as well asenzyme-based histochemical assays), fluorescent, radioactive, andluminescent systems. In one embodiment, the present inventioncontemplates the E. coli β-galactosidase gene (available from PharmaciaBiotech, Pistacataway, N.J.), green fluorescent protein (GFP)(commercially available from Clontech, Palo Alto, Calif.), the humanplacental alkaline phosphatase gene, the chloramphenicolacetyltransferase (CAT) gene; other reporter genes are known to the artand may be employed.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The nucleic acid sequence thus codesfor the amino acid sequence.

The term “heterologous nucleic acid sequence”, as used herein, refers toa nucleotide sequence that is ligated to, or is manipulated to becomeligated to, a nucleic acid sequence to which it is not ligated innature, or to which it is ligated at a different location in nature.Heterologous nucleic acid may include a nucleotide sequence that isnaturally found in the cell into which it is introduced or theheterologous nucleic acid may contain some modification relative to thenaturally occurring sequence.

The term “transgene” refers to any gene coding region, either natural orheterologous nucleic acid sequences or fused homologous or heterologousnucleic acid sequences, introduced into the cells or genome of a testsubject. In the current invention, transgenes are carried on any viralvector that is used to introduce the transgenes to the cells of thesubject.

The term “Second Generation Adenovirus”, as used herein, refers to an Adthat has all or parts of the E1, E2, E3, and, in certain embodiments, E4DNA gene sequences deleted (removed) from the virus.

The term “subject”, as used herein, refers to any animal, e.g., a mammalor marsupial. Subjects of the present invention include but are notlimited to humans, non-human primates (e.g., rhesus or other types ofmacaques), mice, pigs, horses, donkeys, cows, sheep, rats and fowl ofany kind.

Adenovirus Vectors

Compared to First Generation adenovirus vectors, certain embodiments ofthe Second Generation E2b deleted adenovirus vectors of the presentinvention contain additional deletions in the DNA polymerase gene (pol)and deletions of the pre-terminal protein (pTP). E2b deleted vectorshave up to a 13 kb gene-carrying capacity as compared to the 5 to 6 kbcapacity of First Generation adenovirus vectors, easily providing spacefor nucleic acid sequences encoding any of a variety of target antigens,including for example, the Gag, Pol and Nef genes of HIV (Amalfitano, etal. Curr Gene Ther 2/111-133 (2002)). The E2b deleted adenovirus vectorsalso have reduced adverse reactions as compared to First Generationadenovirus vectors (Morral, et al Hum Gene Ther 9/2709-2716 (1998);Hodges, et al. J Gene Med 2/250-259 (2000); DelloRusso, et al. Proc NatlAcad Sci USA 99/12979-12984 (2002); Reddy, et al. Mol Ther 5/63-73(2002); (Amalfitano and Parks, et al. Curr Gene Ther 2/111-133 (2002);Amalfitano Curr Opin Mol Ther 5/362-366 (2003); Everett, et al. HumanGene Ther 14/1715-1726 (2003)) E2b deleted vectors have reducedexpression of viral genes (Hodges, et al. J Gene Med 2/250-259 (2000);Amalfitano, et al. J Virol 72/926-933 (1998); Hartigan-O'Connor, et al.Mol Ther 4/525-533 (2001)), and this characteristic has been reported tolead to extended transgene expression in vivo (Hu, et al. Hum Gene Ther10/355-364 (1999); DelloRusso, et al. Proc Natl Acad Sci USA99/12979-12984 (2002); Reddy, et al. Mol Ther 5/63-73 (2002);(Amalfitano and Parks, et al. Curr Gene Ther 2/111-133 (2002);Amalfitano Curr Opin Mol Ther 5/362-366 (2003); Everett, et al. HumanGene Ther 14/1715-1726 (2003)).

The innate immune response to wild type Ad can be complex, and itappears that Ad proteins expressed from adenovirus vectors play animportant role (Moorhead, et al. J Virol 73/1046-1053 (1999); Nazir, etal. J Investig Med 53/292-304 (2005); Schaack, et al. Proc Natl Acad SciUSA 101/3124-3129 (2004); Schaack, et al. Viral Immunol 18/79-88 (2005);Kiang, et al. Mol Ther 14/588-598 (2006); Hartman, et al. J Virol81/1796-1812 (2007); Hartman, et al. Virology 358/357-372 (2007)).Specifically, the deletions of pre-terminal protein and DNA polymerasein the E2b deleted vectors appear to reduce inflammation during thefirst 24 to 72 hours following injection, whereas First Generationadenovirus vectors stimulate inflammation during this period (Schaack,et al. Proc Natl Acad Sci USA 101/3124-3129 (2004); Schaack, et al.Viral Immunol 18/79-88 (2005); Kiang, et al. Mol Ther 14/588-598 (2006);Hartman, et al. J Virol 81/1796-1812 (2007); Hartman, et al. Virology358/357-372 (2007)). In addition, it has been reported that theadditional replication block created by E2b deletion also leads to a10,000 fold reduction in expression of Ad late genes, well beyond thatafforded by E1, E3 deletions alone (Amalfitano et al. J. Virol.72/926-933 (1998); Hodges et al. J. Gene Med. 2/250-259 (2000)). Thedecreased levels of Ad proteins produced by E2b deleted adenovirusvectors effectively reduce the potential for competitive, undesired,immune responses to Ad antigens, responses that prevent repeated use ofthe platform in Ad immunized or exposed individuals. The reducedinduction of inflammatory response by Second Generation E2b deletedvectors results in increased potential for the vectors to expressdesired vaccine antigens during the infection of antigen presentingcells (i.e. dendritic cells), decreasing the potential for antigeniccompetition, resulting in greater immunization of the vaccine to thedesired antigen relative to identical attempts with First Generationadenovirus vectors. E2b deleted adenovirus vectors provide an improvedAd-based vaccine candidate that is safer, more effective, and moreversatile than previously described vaccine candidates using FirstGeneration adenovirus vectors.

Thus, the present invention contemplates the use of E2b deletedadenovirus vectors, such as those described in U.S. Pat. Nos. 6,063,622;6,451,596; 6,057,158: and 6,083,750. As described in the '622 patent, inorder to further cripple viral protein expression, and also to decreasethe frequency of generating replication competent Ad (RCA), the presentinvention provides adenovirus vectors containing deletions in the E2bregion. Propagation of these E2b deleted adenovirus vectors requirescell lines that express the deleted E2b gene products. The presentinvention also provides such packaging cell lines; for example E.C7(formally called C-7), derived from the HEK-203 cell line (Amalfitano,et al. Proc Natl Acad Sci USA 93/3352-3356 (1996); Amalfitano, et al.Gene Ther 4/258-263 (1997)).

Further, the E2b gene products, DNA polymerase and preterminal protein,can be constitutively expressed in E.C7, or similar cells along with theE1 gene products. Transfer of gene segments from the Ad genome to theproduction cell line has immediate benefits: (1) increased carryingcapacity of the recombinant DNA polymerase and preterminalprotein-deleted adenovirus vector, since the combined coding sequencesof the DNA polymerase and preterminal proteins that can be theoreticallydeleted approaches 4.6 kb; and, (2) a decreased potential of RCAgeneration, since two or more independent recombination events would berequired to generate RCA. Therefore, the E1, Ad DNA polymerase andpreterminal protein expressing cell lines used in the present inventionenable the propagation of adenovirus vectors with a carrying capacityapproaching 13 kb, without the need for a contaminating helper virus[Mitani et al. (1995) Proc. Natl. Acad. Sci. USA 92:3854; Hodges, et al,2000 J Gene Med 2:250-259; (Amalfitano and Parks, Curr Gene Ther2/111-133 (2002)]. In addition, when genes critical to the viral lifecycle are deleted (e.g., the E2b genes), a further crippling of Ad toreplicate or express other viral gene proteins occurs. This willdecrease immune recognition of virally infected cells, and allows forextended durations of foreign transgene expression.

The most important attribute of E1, DNA polymerase, and preterminalprotein deleted vectors, however, is their inability to express therespective proteins from the E1 and E2b regions, as well as a predictedlack of expression of most of the viral structural proteins. Forexample, the major late promoter (MLP) of Ad is responsible fortranscription of the late structural proteins L1 through L5 [Doerfler,In Adenovirus DNA, The Viral Genome and Its Expression (Martinus NijhoffPublishing Boston, 1986)]. Though the MLP is minimally active prior toAd genome replication, the highly toxic Ad late genes are primarilytranscribed and translated from the MLP only after viral genomereplication has occurred [Thomas and Mathews (1980) Cell 22:523]. Thiscis-dependent activation of late gene transcription is a feature of DNAviruses in general, such as in the growth of polyoma and SV-40. The DNApolymerase and preterminal proteins are absolutely required for Adreplication (unlike the E4 or protein IX proteins) and thus theirdeletion is extremely detrimental to adenovirus vector late geneexpression, and the toxic effects of that expression in cells such asAPCs.

In certain embodiments, the adenovirus vectors contemplated for use inthe present invention include E2b deleted adenovirus vectors that have adeletion in the E2b region of the Ad genome and the E1 region but do nothave any other regions of the Ad genome deleted. In another embodiment,the adenovirus vectors contemplated for use in the present inventioninclude E2b deleted adenovirus vectors that have a deletion in the E2bregion of the Ad genome and deletions in the E1 and E3 regions, but noother regions deleted. In a further embodiment, the adenovirus vectorscontemplated for use in the present invention include adenovirus vectorsthat have a deletion in the E2b region of the Ad genome and deletions inthe E1, E3 and partial or complete removal of the E4 regions but noother deletions. In another embodiment, the adenovirus vectorscontemplated for use in the present invention include adenovirus vectorsthat have a deletion in the E2b region of the Ad genome and deletions inthe E1 and E4 regions but no other deletions. In an additionalembodiment, the adenovirus vectors contemplated for use in the presentinvention include adenovirus vectors that have a deletion in the E2a,E2b and E4 regions of the Ad genome but no other deletions. In oneembodiment, the adenovirus vectors for use herein comprise vectorshaving the E1 and DNA polymerase functions of the E2b region deleted butno other deletions. In a further embodiment, the adenovirus vectors foruse herein have the E1 and the preterminal protein functions of the E2bregion deleted and no other deletions. In another embodiment, theadenovirus vectors for use herein have the E1, DNA polymerase and thepreterminal protein functions deleted, and no other deletions. In oneparticular embodiment, the adenovirus vectors contemplated for useherein are deleted for at least a portion of the E2b region and the E1region, but are not “gutted” adenovirus vectors. In this regard, thevectors may be deleted for both the DNA polymerase and the preterminalprotein functions of the E2b region. In an additional embodiment, theadenovirus vectors for use in the present invention include adenovirusvectors that have a deletion in the E1, E2b and 100K regions of theadenovirus genome. In one embodiment, the adenovirus vectors for useherein comprise vectors having the E1, E2b and protease functionsdeleted but no other deletions. In a further embodiment, the adenovirusvectors for use herein have the E1 and the E2b regions deleted, whilethe fiber genes have been modified by mutation or other alterations (forexample to alter Ad tropism). Removal of genes from the E3 or E4 regionsmay be added to any of the mentioned adenovirus vectors. In certainembodiments, the adenovirus vector may be a “gutted” adenovirus vector.

The term “E2b deleted”, as used herein, refers to a specific DNAsequence that is mutated in such a way so as to prevent expressionand/or function of at least one E2b gene product. Thus, in certainembodiments, “E2b deleted” refers to a specific DNA sequence that isdeleted (removed) from the Ad genome. E2b deleted or “containing adeletion within the E2b region” refers to a deletion of at least onebase pair within the E2b region of the Ad genome. Thus, in certainembodiments, more than one base pair is deleted and in furtherembodiments, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, or 150 base pairs are deleted. In another embodiment, thedeletion is of more than 150, 160, 170, 180, 190, 200, 250, or 300 basepairs within the E2b region of the Ad genome. An E2b deletion may be adeletion that prevents expression and/or function of at least one E2bgene product and therefore, encompasses deletions within exons encodingportions of E2b-specific proteins as well as deletions within promoterand leader sequences. In certain embodiments, an E2b deletion is adeletion that prevents expression and/or function of one or both of theDNA polymerase and the preterminal protein of the E2b region. In afurther embodiment, “E2b deleted” refers to one or more point mutationsin the DNA sequence of this region of an Ad genome such that one or moreencoded proteins is non-functional. Such mutations include residues thatare replaced with a different residue leading to a change in the aminoacid sequence that result in a nonfunctional protein.

As would be understood by the skilled artisan upon reading the presentdisclosure, other regions of the Ad genome can be deleted. Thus to be“deleted” in a particular region of the Ad genome, as used herein,refers to a specific DNA sequence that is mutated in such a way so as toprevent expression and/or function of at least one gene product encodedby that region. In certain embodiments, to be “deleted” in a particularregion refers to a specific DNA sequence that is deleted (removed) fromthe Ad genome in such a way so as to prevent the expression and/or thefunction encoded by that region (e.g., E2b functions of DNA polymeraseor preterminal protein function). “Deleted” or “containing a deletion”within a particular region refers to a deletion of at least one basepair within that region of the Ad genome. Thus, in certain embodiments,more than one base pair is deleted and in further embodiments, at least20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 basepairs are deleted from a particular region. In another embodiment, thedeletion is more than 150, 160, 170, 180, 190, 200, 250, or 300 basepairs within a particular region of the Ad genome. These deletions aresuch that expression and/or function of the gene product encoded by theregion is prevented. Thus deletions encompass deletions within exonsencoding portions of proteins as well as deletions within promoter andleader sequences. In a further embodiment, “deleted” in a particularregion of the Ad genome refers to one or more point mutations in the DNAsequence of this region of an Ad genome such that one or more encodedproteins is non-functional. Such mutations include residues that arereplaced with a different residue leading to a change in the amino acidsequence that result in a nonfunctional protein.

The deleted adenovirus vectors of the present invention can be generatedusing recombinant techniques known in the art (see e.g., Amalfitano etal., 1998 J. Virol. 72:926-933; Hodges, et al., 2000 J Gene Med2:250-259).

As would be recognized by the skilled artisan, the adenovirus vectorsfor use in the present invention can be successfully grown to hightiters using an appropriate packaging cell line that constitutivelyexpresses E2b gene products and products of any of the necessary genesthat may have been deleted. In certain embodiments, HEK-293-derivedcells that not only constitutively express the E1 and DNA polymeraseproteins, but also the Ad-preterminal protein, can be used. In oneembodiment, E.C7 cells are used to successfully grow high titer stocksof the adenovirus vectors (see e.g., Amalfitano et al., J. Virol. 199872:926-933; Hodges, et al. J Gene Med 2/250-259 (2000))

In order to delete critical genes from self-propagating adenovirusvectors, the proteins encoded by the targeted genes have to first becoexpressed in HEK-293 cells, or similar, along with the E1 proteins.Therefore, only those proteins which are non-toxic when coexpressedconstitutively (or toxic proteins inducibly-expressed) can be utilized.Coexpression in HEK-293 cells of the E1 and E4 genes has beendemonstrated (utilizing inducible, not constitutive, promoters) [Yeh etal. (1996) J. Virol. 70:559; Wang et al. (1995) Gene Therapy 2:775; andGorziglia et al. (1996) J. Virol. 70:4173]. The E1 and protein IX genes(a virion structural protein) have been coexpressed [Caravokyri andLeppard (1995) J. Virol. 69:6627], and coexpression of the E1, E4, andprotein IX genes has also been described [Krougliak and Graham (1995)Hum. Gene Ther. 6:1575]. The E1 and 100 k genes have been successfullyexpressed in transcomplementing cell lines, as have E1 and proteasegenes (Oualikene, et al. Hum Gene Ther 11/1341-1353 (2000); Hodges, etal. J. Virol 75/5913-5920 (2001)).

Cell lines coexpressing E1 and E2b gene products for use in growing hightiters of E2b deleted Ad particles are described in U.S. Pat. No.6,063,622. The E2b region encodes the viral replication proteins whichare absolutely required for Ad genome replication [Doerfler, supra andPronk et al. (1992) Chromosoma 102:S39-S45]. Useful cell linesconstitutively express the approximately 140 kD Ad-DNA polymerase and/orthe approximately 90 kD preterminal protein. In particular, cell linesthat have high-level, constitutive coexpression of the E1, DNApolymerase, and preterminal proteins, without toxicity (e.g. E.C7), aredesirable for use in propagating Ad for use in multiple vaccinations.These cell lines permit the propagation of adenovirus vectors deletedfor the E1, DNA polymerase, and preterminal proteins.

The recombinant Ad of the present invention can be propagated usingtechniques known in the art. For example, in certain embodiments, tissueculture plates containing E.C7 cells are infected with the adenovirusvector virus stocks at an appropriate MOI (e.g., 5) and incubated at37.0° C. for 40-96 h. The infected cells are harvested, resuspended in10 mM Tris-CI (pH 8.0), and sonicated, and the virus is purified by tworounds of cesium chloride density centrifugation. In certain techniques,the virus containing band is desalted over a Sephadex CL-6B column(Pharmacia Biotech, Piscataway, N.J.), sucrose or glycerol is added, andaliquots are stored at −80° C. In some embodiments, the virus will beplaced in a solution designed to enhance its stability, such as A195(Evans, et al. J Pharm Sci 93/2458-2475 (2004)) The titer of the stockis measured (e.g., by measurement of the optical density at 260 nm of analiquot of the virus after SDS lysis). In another embodiment, plasmidDNA, either linear or circular, encompassing the entire recombinant E2bdeleted adenovirus vector can be transfected into E.C7, or similarcells, and incubated at 37.0° C. until evidence of viral production ispresent (e.g. the cytopathic effect). The conditioned media from thesecells can then be used to infect more E.C7, or similar cells, to expandthe amount of virus produced, before purification. Purification can beaccomplished by two rounds of cesium chloride density centrifugation orselective filtration. In certain embodiments, the virus may be purifiedby column chromatography, using commercially available products (e.g.Adenopure from Puresyn, Inc., Malvern, Pa.) or custom madechromatographic columns.

Generally, the recombinant Ad of the present invention comprises enoughof the virus to ensure that the cells to be infected are confronted witha certain number of viruses. Thus, the present invention provides astock of recombinant Ad, preferably an RCA-free stock of recombinant Ad.The preparation and analysis of Ad stocks is well known in the art.Viral stocks vary considerably in titer, depending largely on viralgenotype and the protocol and cell lines used to prepare them. The viralstocks of the present invention can have a titer of at least about 10⁶,10⁷, or 10⁸ pfu/ml, and many such stocks can have higher titers, such asat least about 10⁹, 10¹⁰, 10¹¹, or 10¹² pfu/ml. Depending on the natureof the recombinant virus and the packaging cell line, it is possiblethat a viral stock of the present invention can have a titer of evenabout 10¹³ particles/ml or higher.

Further information on viral delivery systems is known in the art andcan be found, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci.USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103,1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112,4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627,1988; Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., Proc.Natl. Acad. Sci. USA 91:215-219, 1994; Kass-Eisler et al., Proc. Natl.Acad. Sci. USA 90:11498-11502, 1993; Guzman et al., Circulation88:2838-2848, 1993; and Guzman et al., Cir. Res. 73:1202-1207, 1993.

Heterologous Nucleic Acid

The adenovirus vectors of the present invention also compriseheterologous nucleic acid sequences that encode one or more targetantigens of interest, or variants, fragments or fusions thereof, againstwhich it is desired to generate an immune response. In some embodiments,the adenovirus vectors of the present invention comprise heterologousnucleic acid sequences that encode one or more proteins, variantsthereof, fusions thereof, or fragments thereof, that can modulate theimmune response. In a further embodiment of the invention, theadenovirus vector of the present invention encodes one or moreantibodies against specific antigens, such as anthrax protectiveantigen, permitting passive immunotherapy. In certain embodiments, theadenovirus vectors of the present invention comprise heterologousnucleic acid sequences encoding one or more proteins having therapeuticeffect (e.g., anti-viral, anti-bacterial, anti-parasitic, or anti-tumorfunction). Thus the present invention provides the Second Generation E2bdeleted adenovirus vectors that comprise a heterologous nucleic acidsequence.

As such, the present invention further provides nucleic acid sequences,also referred to herein as polynucleotides, that encode one or moretarget antigens of interest, or fragments or variants thereof. As such,the present invention provides polynucleotides that encode targetantigens from any source as described further herein, vectors comprisingsuch polynucleotides and host cells transformed or transfected with suchexpression vectors. The terms “nucleic acid” and “polynucleotide” areused essentially interchangeably herein. As will be also recognized bythe skilled artisan, polynucleotides of the invention may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. RNA molecules may includeHnRNA molecules, which contain introns and correspond to a DNA moleculein a one-to-one manner, and mRNA molecules, which do not containintrons. Additional coding or non-coding sequences may, but need not, bepresent within a polynucleotide of the present invention, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials. An isolated polynucleotide, as used herein, meansthat a polynucleotide is substantially away from other coding sequences.For example, an isolated DNA molecule as used herein does not containlarge portions of unrelated coding DNA, such as large chromosomalfragments or other functional genes or polypeptide coding regions. Ofcourse, this refers to the DNA molecule as originally isolated, and doesnot exclude genes or coding regions later added to the segmentrecombinantly in the laboratory.

As will be understood by those skilled in the art, the polynucleotidesof this invention can include genomic sequences, extra-genomic andplasmid-encoded sequences and smaller engineered gene segments thatexpress, or may be adapted to express target antigens as describedherein, fragments of antigens, peptides and the like. Such segments maybe naturally isolated, or modified synthetically by the hand of man.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a target antigen polypeptide/protein/epitope ofthe invention or a portion thereof) or may comprise a sequence thatencodes a variant, fragment, or derivative of such a sequence. Incertain embodiments, the polynucleotide sequences set forth hereinencode target antigen proteins as described herein. In some embodiments,polynucleotides represent a novel gene sequence that has been optimizedfor expression in specific cell types (i.e. human cell lines) that maysubstantially vary from the native nucleotide sequence or variant butencode a similar protein antigen.

In other related embodiments, the present invention providespolynucleotide variants having substantial identity to native sequencesencoding proteins (e.g., target antigens of interest) as describedherein, for example those comprising at least 70% sequence identity,preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% orhigher, sequence identity compared to a native polynucleotide sequenceencoding the polypeptides of this invention using the methods describedherein, (e.g., BLAST analysis using standard parameters, as describedbelow). One skilled in this art will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning and thelike.

Typically, polynucleotide variants will contain one or moresubstitutions, additions, deletions and/or insertions, preferably suchthat the immunogenicity of the epitope of the polypeptide encoded by thevariant polynucleotide or such that the immunogenicity of theheterologous target protein is not substantially diminished relative toa polypeptide encoded by the native polynucleotide sequence. Asdescribed elsewhere herein, the polynucleotide variants preferablyencode a variant of the target antigen, or a fragment (e.g., an epitope)thereof wherein the propensity of the variant polypeptide or fragment(e.g., epitope) thereof to react with antigen-specific antisera and/orT-cell lines or clones is not substantially diminished relative to thenative polypeptide. The term “variants” should also be understood toencompass homologous genes of xenogenic origin. In particularembodiments, variants or fragments of target antigens are modified suchthat they have one or more reduced biological activities. For example,an oncogenic protein target antigen may be modified to reduce oreliminate the oncogenic activity of the protein, or a viral protein maybe modified to reduce or eliminate one or more activities or the viralprotein. An example of a modified HER2 protein is a kinase-inactive HER2having a K753A mutation that renders it non-oncogenic, as described inMorse, M. A. et al., Int. J. Cancer, 2009, Vol. 9999, Issue 999A, pageNA, published online 23 Oct. 2009; and Akiyama, T. et al., Mol. Cell.Biol. 1991, 11(2):833-842.

The present invention provides polynucleotides that comprise or consistof at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 11, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 or morecontiguous nucleotides encoding a polypeptide, including target proteinantigens, as described herein, as well as all intermediate lengths therebetween. It will be readily understood that “intermediate lengths”, inthis context, means any length between the quoted values, such as 16,17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53,etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including allintegers through 200-500; 500-1,000, and the like. A polynucleotidesequence as described herein may be extended at one or both ends byadditional nucleotides not found in the native sequence encoding apolypeptide as described herein, such as an epitope or heterologoustarget protein. This additional sequence may consist of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides ormore, at either end of the disclosed sequence or at both ends of thedisclosed sequence.

The polynucleotides of the present invention, or fragments thereof,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, expression controlsequences, polyadenylation signals, additional restriction enzyme sites,multiple cloning sites, other coding segments, and the like, such thattheir overall length may vary considerably. It is therefore contemplatedthat a nucleic acid fragment of almost any length may be employed, withthe total length preferably being limited by the ease of preparation anduse in the intended recombinant DNA protocol. For example, illustrativepolynucleotide segments with total lengths of about 1000, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000, 10,000, about 500, about 200, about100, about 50 base pairs in length, and the like, (including allintermediate lengths) are contemplated to be useful in manyimplementations of this invention.

When comparing polynucleotide sequences, two sequences are said to be“identical” if the sequence of nucleotides in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., Unified Approach to Alignment and Phylogenes, pp. 626-645 (1990);Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., CABIOS 5:151-153 (1989); Myers, E. W.and Muller W., CABIOS 4:11-17 (1988); Robinson, E. D., Comb. Theor11:105 (1971); Saitou, N. Nei, M., Mol. Biol. Evol. 4:406-425 (1987);Sneath, P. H. A. and Sokal, R. R., Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.(1973); Wilbur, W. J. and Lipman, D. J., Proc. Natl. Acad., Sci. USA80:726-730 (1983).

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman, Add.APL. Math 2:482 (1981), by the identity alignment algorithm of Needlemanand Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similaritymethods of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444(1988), by computerized implementations of these algorithms (GAP,BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.),or by inspection.

One example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nucl. Acids Res.25:3389-3402 (1977), and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 can be used, for example withthe parameters described herein, to determine percent sequence identityfor the polynucleotides of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information. In one illustrative example, cumulativescores can be calculated using, for nucleotide sequences, the parametersM (reward score for a pair of matching residues; always >0) and N(penalty score for mismatching residues; always <0). Extension of theword hits in each direction are halted when: the cumulative alignmentscore falls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T and X determinethe sensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, andexpectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff andHenikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments, (B) of50, expectation (E) of 10, M=5, N=−4 and a comparison of both strands.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12percent, as compared to the reference sequences (which does not compriseadditions or deletions) for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid bases occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the reference sequence (i.e., thewindow size) and multiplying the results by 100 to yield the percentageof sequence identity.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a particular antigen of interest, or fragmentthereof, as described herein. Some of these polynucleotides bear minimalhomology to the nucleotide sequence of any native gene. Nonetheless,polynucleotides that vary due to differences in codon usage arespecifically contemplated by the present invention. Further, alleles ofthe genes comprising the polynucleotide sequences provided herein arewithin the scope of the present invention. Alleles are endogenous genesthat are altered as a result of one or more mutations, such asdeletions, additions and/or substitutions of nucleotides. The resultingmRNA and protein may, but need not, have an altered structure orfunction. Alleles may be identified using standard techniques (such ashybridization, amplification and/or database sequence comparison).

Therefore, in another embodiment of the invention, a mutagenesisapproach, such as site-specific mutagenesis, is employed for thepreparation of variants and/or derivatives of the target antigensequences, or fragments thereof, as described herein. By this approach,specific modifications in a polypeptide sequence can be made throughmutagenesis of the underlying polynucleotides that encode them. Thesetechniques provide a straightforward approach to prepare and testsequence variants, for example, incorporating one or more of theforegoing considerations, by introducing one or more nucleotide sequencechanges into the polynucleotide.

Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Mutations may be employed in aselected polynucleotide sequence to improve, alter, decrease, modify, orotherwise change the properties of the polynucleotide itself, and/oralter the properties, activity, composition, stability, or primarysequence of the encoded polypeptide.

In certain embodiments of the present invention, the inventorscontemplate the mutagenesis of the disclosed polynucleotide sequences toalter one or more properties of the encoded polypeptide, such as theimmunogenicity of an epitope comprised in a polypeptide or theoncogenicity of a target antigen. In certain embodiments, a kinasedomain is inactivated in a target antigen. Assays to test theimmunogenicity of a polypeptide or variant thereof are well known in theart and include, but are not limited to, T cell cytotoxicity assays(CTL/chromium release assays), T cell proliferation assays,intracellular cytokine staining, ELISA, ELISpot, etc. The techniques ofsite-specific mutagenesis are well known in the art, and are widely usedto create variants of both polypeptides and polynucleotides. Forexample, site-specific mutagenesis is often used to alter a specificportion of a DNA molecule. In such embodiments, a primer comprisingtypically about 14 to about 25 nucleotides or so in length is employed,with about 5 to about 10 residues on both sides of the junction of thesequence being altered.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis provides a means ofproducing potentially useful species and is not meant to be limiting, asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.Specific details regarding these methods and protocols are found in theteachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991;Kuby, 1994; and Maniatis et al., 1982.

Polynucleotide segments or fragments encoding the polypeptides of thepresent invention may be readily prepared by, for example, directlysynthesizing the fragment by chemical means, as is commonly practicedusing an automated oligonucleotide synthesizer. Also, fragments may beobtained by application of nucleic acid reproduction technology, such asthe PCR™ technology of U.S. Pat. No. 4,683,202, by introducing selectedsequences into recombinant vectors for recombinant production, and byother recombinant DNA techniques generally known to those of skill inthe art of molecular biology (see for example, Current Protocols inMolecular Biology, John Wiley and Sons, NY, N.Y.).

In order to express a desired target antigen polypeptide or fragment orvariant thereof, or fusion protein comprising any of the above, asdescribed herein, the nucleotide sequences encoding the polypeptide, orfunctional equivalents, are inserted into an appropriate Ad as describedelsewhere herein using recombinant techniques known in the art. Theappropriate adenovirus vector contains the necessary elements for thetranscription and translation of the inserted coding sequence and anydesired linkers. Methods which are well known to those skilled in theart may be used to construct these adenovirus vectors containingsequences encoding a polypeptide of interest and appropriatetranscriptional and translational control elements. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination. Such techniques are described, forexample, in Amalfitano et al., 1998 J. Virol. 72:926-933; Hodges, etal., 2000 J Gene Med 2:250-259; Sambrook, J. et al. (1989) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.,and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology,John Wiley & Sons, New York. N.Y.

A variety of vector/host systems may be utilized to contain and producepolynucleotide sequences. These include, but are not limited to,microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA vectors; yeast transformed withyeast vectors; insect cell systems infected with virus vectors (e.g.,baculovirus); plant cell systems transformed with virus vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or withbacterial vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The “control elements” or “regulatory sequences” present in anadenovirus vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, sequences encoding apolypeptide of interest may be ligated into an Adtranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing the polypeptide in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf, D. et al.(1994) Results Probl. Cell Differ. 20:125-162). Specific terminationsequences, either for transcription or translation, may also beincorporated in order to achieve efficient translation of the sequenceencoding the polypeptide of choice.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products (e.g., target antigens of interest),using either polyclonal or monoclonal antibodies specific for theproduct are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson a given polypeptide may be preferred for some applications, but acompetitive binding assay may also be employed. These and other assaysare described, among other places, in Hampton, R. et al. (1990;Serological Methods, a Laboratory Manual, APS Press, St Paul. Minn.) andMaddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).

The adenovirus vectors of the present invention comprise nucleic acidsequences encoding one or more antigens of interest, or variants orfragments thereof. The nucleic acid sequence may also contain a productthat can be detected or selected for. As referred to herein, a“reporter” gene is one whose product can be detected, such as byfluorescence, enzyme activity on a chromogenic or fluorescent substrate,and the like or selected for by growth conditions. Such reporter genesinclude, without limitation, green fluorescent protein (GFP),β-galactosidase, chloramphenicol acetyltransferase (CAT), luciferase,neomycin phosphotransferase, secreted alkaline phosphatase (SEAP), andhuman growth hormone (HGH). Selectable markers include drug resistances,such as neomycin (G418), hygromycin, and the like.

The nucleic acid encoding an antigen of interest may also comprise apromoter or expression control sequence. This is a nucleic acid-sequencethat controls expression of the nucleic acid sequence encoding a targetantigen and generally is active or activatable in the targeted cell. Thechoice of the promoter will depend in part upon the targeted cell typeand the degree or type of control desired. Promoters that are suitablewithin the context of the present invention include, without limitation,constitutive, inducible, tissue specific, cell type specific, temporalspecific, or event-specific.

Examples of constitutive or nonspecific promoters include the SV40 earlypromoter (U.S. Pat. No. 5,118,627), the SV40 late promoter (U.S. Pat.No. 5,118,627), CMV early gene promoter (U.S. Pat. No. 5,168,062),bovine papilloma virus promoter, and adenovirus promoter. In addition toviral promoters, cellular promoters are also amenable within the contextof this invention. In particular, cellular promoters for the so-calledhousekeeping genes are useful (e.g., β-actin). Viral promoters aregenerally stronger promoters than cellular promoters.

Inducible promoters may also be used. These promoters include MMTV LTR(PCT WO 91/13160), inducible by dexamethasone, metallothionein,inducible by heavy metals, and promoters with cAMP response elements,inducible by cAMP, heat shock, promoter. By using an inducible promoter,the nucleic acid may be delivered to a cell and will remain quiescentuntil the addition of the inducer. This allows further control on thetiming of production of the protein of interest.

Event-type specific promoters are active or upregulated only upon theoccurrence of an event, such as tumorigenicity or viral infection, forexample. The HIV LTR is a well-known example of an event-specificpromoter. The promoter is inactive unless the tat gene product ispresent, which occurs upon viral infection. Some event-type promotersare also tissue-specific. Preferred event-type specific promotersinclude promoters activated upon viral infection.

Examples of promoters discussed herein include, but are not limited to,promoters for alphafetoprotein, alpha actin, myo D, carcinoembryonicantigen, VEGF-receptor (GenBank Accession No. X89776); FGF receptor; TEKor tie 2 (GenBank Accession No. L06139); tie (GenBank Accession Nos.X60954; S89716); urokinase receptor (GenBank Accession No. S78532); E-and P-selectins (GenBank Accession Nos. M64485; L01874); VCAM-1 (GenBankAccession No. M92431); endoglin (GenBank Accession No. HSENDOG);endosialin (Rettig et al., PNAS 89:10832, 1992); alpha V-beta3 integrin(Villa-Garcia et al., Blood 3:668, 1994; Donahue et al., BBA 1219:228,1994); endothelin-1 (GenBank Accession Nos. M25377; J04819; J05489);ICAM-3 (GenBank Accession No. S50015); E9 antigen (Wang et al., Int. J.Cancer 54:363, 1993); von Willebrand factor (GenBank Accession Nos.HUMVWFI. HUMVWFA); CD44 (GenBank Accession No. HUMCD44B); CD40 (GenBankAccession Nos. HACD40L; HSCD405FR); vascular-endothelial cadherin(Martin-Padura et al., J. Pathol. 175:51, 1995); notch 4 (Uyttendaele etal., Development 122:2251, 1996) high molecular weightmelanoma-associated antigen; prostate specific antigen-1, probasin, FGFreceptor, VEGF receptor, erb B2; erb B3; erb B4; MUC-1; HSP-27; int-1;int-2, CEA, HBEGF receptor; EGF receptor; tyrosinase, MAGE, IL-2receptor; prostatic acid phosphatase, probasin, prostate specificmembrane antigen, alpha-crystallin, PDGF receptor, integrin receptor,α-actin, SM1 and SM2 myosin heavy chains, calponin-h1, SM22 alphaangiotensin receptor, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, immunoglobulin heavy chain,immunoglobulin light chain, CD4, and the like are useful within thecontext of this invention.

In addition to the promoter, repressor sequences, negative regulators,or tissue-specific silencers may be inserted to reduce non-specificexpression of the polynucleotide. Multiple repressor elements may beinserted in the promoter region. Repression of transcription isindependent of the orientation of repressor elements or distance fromthe promoter. One type of repressor sequence is an insulator sequence.Such sequences inhibit transcription (Dunaway et al., Mol Cell Biol 17:182-9, 1997; Gdula et al., Proc Natl Acad Sci USA 93:9378-83, 1996, Chanet al., J Virol 70: 5312-28, 1996; Scott and Geyer, EMBO J 14: 6258-67,1995; Kalos and Fournier, Mol Cell Biol 15: 198-207, 1995; Chung et al.,Cell 74: 505-14, 1993) and will silence background transcription.

Negative regulatory elements have been characterized in the promoterregions of a number of different genes. The repressor element functionsas a repressor of transcription in the absence of factors, such assteroids, as does the NSE in the promoter region of the ovalbumin gene(Haecker et al., Mol. Endocrinology 9:1113-1126, 1995). These negativeregulatory elements bind specific protein complexes from oviduct, noneof which are sensitive to steroids. Three different elements are locatedin the promoter of the ovalbumin gene. Oligonucleotides corresponding toportions of these elements repress viral transcription of the TKreporter. One of the silencer elements shares sequence identity withsilencers in other genes (TCTCTCCNA).

Repressor elements have also been identified in the promoter region of avariety of genes, including the collagen II gene, for example. Gelretardation studies showed that nuclear factors from HeLa cells bindspecifically to DNA fragments containing the silencer region, whereaschondrocyte nuclear extracts did not show any binding activity (Savangeret al., J. Biol. Chem. 265(12):6669-6674, 1990). Repressor elements havealso been shown to regulate transcription in the carbamyl phosphatesynthetase gene (Goping et al., Nucleic Acid Research 23(10):1717-1721,1995). This gene is expressed in only two different cell types,hepatocytes and epithelial cells of the intestinal mucosa. Negativeregulatory regions have also been identified in the promoter region ofthe choline acetyltransferase gene, the albumin promoter (Hu et al., J.Cell Growth Differ. 3(9):577-588, 1992), phosphoglycerate kinase (PGK-2)gene promoter (Misuno et al., Gene 119(2):293-297, 1992), and in the6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase gene, in which thenegative regulatory element inhibits transcription in non-hepatic celllines (Lemaigre et al., Mol. Cell Biol. 11(2):1099-1106). Furthermore,the negative regulatory element Tse-1 has been identified in a number ofliver specific genes, including tyrosine aminotransferase (TAT). TATgene expression is liver specific and inducible by both glucocorticoidsand the cAMP signaling pathway. The cAMP response element (CRE) has beenshown to be the target for repression by Tse-1 and hepatocyte-specificelements (Boshart et al., Cell 61(5):905-916, 1990). Accordingly, it isclear that varieties of such elements are known or are readilyidentified.

In certain embodiments, elements that increase the expression of thedesired target antigen are incorporated into the nucleic acid sequenceof the adenovirus vectors described herein. Such elements includeinternal ribosome binding sites (IRES; Wang and Siddiqui, Curr. Top.Microbiol. Immunol 203:99, 1995; Ehrenfeld and Semler, Curr. Top.Microbiol. Immunol. 203:65, 1995; Rees et al., Biotechniques 20:102,1996; Sugimoto et al., Biotechnology 12:694, 1994). IRES increasetranslation efficiency. As well, other sequences may enhance expression.For some genes, sequences especially at the 5′ end inhibit transcriptionand/or translation. These sequences are usually palindromes that canform hairpin structures. Any such sequences in the nucleic acid to bedelivered are generally deleted. Expression levels of the transcript ortranslated product are assayed to confirm or ascertain which sequencesaffect expression. Transcript levels may be assayed by any known method,including Northern blot hybridization, RNase probe protection and thelike. Protein levels may be assayed by any known method, includingELISA.

As would be recognized by the skilled artisan, the adenovirus vectors ofthe present invention comprising heterologous nucleic acid sequences canbe generated using recombinant techniques known in the art, such asthose described in Maione et al., 2001 Proc Natl Acad Sci USA,98:5986-5991; Maione et al., 2000 Hum Gene Ther 11:859-868; Sandig etal. 2000 Proc Natl Acad Sci USA, 97:1002-1007; Harui et al. 2004 GeneTherapy, 11:1617-1626; Parks et al., 1996 Proc Natl Acad Sci USA,93:13565-13570; DelloRusso et al., 2002 Proc Natl Acad Sci USA,99:12979-12984; Current Protocols in Molecular Biology, John Wiley andSons, NY, N.Y.).

As noted above, the adenovirus vectors of the present invention comprisenucleic acid sequences that encode one or more target proteins orantigens of interest. In this regard, the vectors may contain nucleicacid encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or more different target antigens of interest. The targetantigens may be a full length protein or may be a fragment (e.g., anepitope) thereof. The adenovirus vectors may contain nucleic acidsequences encoding multiple fragments or epitopes from one targetprotein of interest or may contain one or more fragments or epitopesfrom numerous different target proteins of interest.

The term “target antigen” or “target protein” as used herein refers to amolecule, such as a protein, against which an immune response is to bedirected. The target antigen may comprise any substance against which itis desirable to generate an immune response but generally, the targetantigen is a protein. A target antigen may comprise a full lengthprotein or a fragment thereof that induces an immune response (i.e., animmunogenic fragment). A target antigen or fragment thereof may bemodified, e.g., to reduce one or more biological activities of thetarget antigen or to enhance its immunogenicity.

An “immunogenic fragment,” as used herein is a fragment of a polypeptidethat is specifically recognized (i.e., specifically bound) by a B-celland/or T-cell surface antigen receptor resulting in the generation of animmune response specifically against the fragment. In certainembodiments, immunogenic fragments bind to an MHC class I or class IImolecule. As used herein, an immunogenic fragment is said to “bind to”an MHC class I or class II molecule if such binding is detectable usingany assay known in the art. For example, the ability of a polypeptide tobind to MHC class I may be evaluated indirectly by monitoring theability to promote incorporation of ¹²⁵I labeled β2-microglobulin (β2m)into MHC class I/β2m/peptide heterotrimeric complexes (see Parker etal., J. Immunol. 152:163, 1994). Alternatively, functional peptidecompetition assays that are known in the art may be employed.Immunogenic fragments of polypeptides may generally be identified usingwell known techniques, such as those summarized in Paul, FundamentalImmunology, 3rd ed., 243-247 (Raven Press, 1993) and references citedtherein. Representative techniques for identifying immunogenic fragmentsinclude screening polypeptides for the ability to react withantigen-specific antisera and/or T-cell lines or clones. An immunogenicfragment of a particular target polypeptide is a fragment that reactswith such antisera and/or T-cells at a level that is not substantiallyless than the reactivity of the full length target polypeptide (e.g., inan ELISA and/or T-cell reactivity assay). In other words, an immunogenicfragment may react within such assays at a level that is similar to orgreater than the reactivity of the full length polypeptide. Such screensmay generally be performed using methods well known to those of ordinaryskill in the art, such as those described in Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.

Target antigens of the present invention include but are not limited toantigens derived from any of a variety of infectious agents or cancercells. As used herein, an “infectious agent” is any living organismcapable of infecting a host and “cancer” means a neoplastic cell.Infectious agents include, for example, bacteria, any variety ofviruses, such as, single stranded RNA viruses, single stranded DNAviruses, fungi, parasites, and protozoa. Examples of infectious agentsinclude, but are not limited to, Actinobacillus spp., Actinomyces spp.,Adenovirus (types 1, 2, 3, 4, 5 et 7), Adenovirus (types 40 and 41),Aerococcus spp., Aeromonas hydrophila, Ancylostoma duodenale,Angiostrongylus cantonensis, Ascaris lumbricoides, Ascaris spp.,Aspergillus spp., Babesia spp, B. microti, Bacillus anthracis, Bacilluscereus, Bacteroides spp., Balantidium coli, Bartonella bacilliformis,Blastomyces dermatitidis, Bluetongue virus, Bordetella bronchiseptica,Bordetella pertussis, Borrelia afzelii, Borrelia burgdorferi, Borreliagarinii, Branhamella catarrhalis, Brucella spp. (B. abortus, B. canis,B. melitensis, B. suis), Brugia spp., Burkholderia, (Pseudomonas)mallei, Burkholderia (Pseudomonas) pseudomallei, California serogroup,Campylobacter fetus subsp. Fetus, Campylobacter jejuni, C. coli, C.fetus subsp. Jejuni, Candida albicans, Capnocytophaga spp., Chikungunyavirus, Chlamydia psittaci, Chlamydia trachomatis, Citrobacter spp.,Clonorchis sinensis, Clostridium botulinum, Clostridium difficile,Clostridium perfringens, Clostridium tetani, Clostridium spp. (with theexception of those species listed above), Coccidioides immitis, Coloradotick fever virus, Corynebacterium diphtheriae, Coxiella burnetii,Coxsackievirus, Creutzfeldt-Jakob agent, Kuru agent, Crimean-Congohemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidiumparvum, Cytomegalovirus, Cyclospora cayatanesis, Dengue virus (1, 2, 3,4), Diphtheroids, Eastern (Western) equine encephalitis virus, Ebolavirus, Echinococcus granulosus, Echinococcus multilocularis, Echovirus,Edwardsiella tarda, Entamoeba histolytica, Enterobacter spp.,Enterovirus 70, Epidermophyton floccosum, Ehrlichia spp, Ehrlichiasennetsu, Microsporum spp. Trichophyton spp., Epstein-Barr virus,Escherichia coli, enterohemorrhagic, Escherichia coli, enteroinvasive,Escherichia coli, enteropathogenic, Escherichia coli, enterotoxigenic,Fasciola hepatica, Francisella tularensis, Fusobacterium spp., Gemellahaemolysans, Giardia lamblia, Guanarito virus, Haemophilus ducreyi,Haemophilus influenzae (group b), Hantavirus, Hepatitis A virus,Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis Evirus, Herpes simplex virus, Herpesvirus simiae, Histoplasma capsulatum,Human coronavirus, Human immunodeficiency virus, Human papillomavirus,Human rotavirus, Human T-lymphotrophic virus, Influenza virus includingH5N1, Junin virus/Machupo virus, Klebsiella spp., Kyasanur Forestdisease virus, Lactobacillus spp., Lassa virus, Legionella pneumophila,Leishmania major, Leishmania infantum, Leishmania spp., Leptospirainterrogans, Listeria monocytogenes, Lymphocytic choriomeningitis virus,Machupo virus, Marburg virus, Measles virus, Micrococcus spp., Moraxellaspp., Mycobacterium spp. (other than M. bovis, M. tuberculosis, M.avium, M. leprae), Mycobacterium tuberculosis, M. bovis, Mycoplasmahominis, M. orale, M. salivarium, M. fermentans, Mycoplasma pneumoniae,Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseriameningitides, Neisseria spp. (other than N. gonorrhoeae and N.meningitidis), Nocardia spp., Norwalk virus, Omsk hemorrhagic fevervirus, Onchocerca volvulus, Opisthorchis spp., Parvovirus B19,Pasteurella spp., Peptococcus spp., Peptostreptococcus spp., Plasmodiumfalciparum, Plasmodium vivax, Plasmodium spp., Plesiomonas shigelloides,Powassan encephalitis virus, Proteus spp., Pseudomonas spp. (other thanP. mallei, P. pseudomallei), Rabies virus, Respiratory syncytial virus,Rhinovirus, Rickettsia akari, Rickettsia prowazekii, R. Canada,Rickettsia rickettsii, Rift Valley virus, Ross river virus/O'Nyong-Nyongvirus, Rubella virus, Salmonella choleraesuis, Salmonella paratyphi,Salmonella typhi, Salmonella spp. (with the exception of those specieslisted above), Schistosoma spp., Scrapie agent, Serratia spp., Shigellaspp., Sindbis virus, Sporothrix schenckii, St. Louis encephalitis virus,Murray Valley encephalitis virus, Staphylococcus aureus, Streptobacillusmoniliformis, Streptococcus agalactiae, Streptococcus faecalis,Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcussalivarius, Taenia saginata, Taenia solium, Toxocara canis, T. cati, T.cruzi, Toxoplasma gondii, Treponema pallidum, Trichinella spp.,Trichomonas vaginalis, Trichuris trichiura, Trypanosoma brucei,Trypanosoma cruzi, Ureaplasma urealyticum, Vaccinia virus,Varicella-zoster virus, eastern equine encephalitis virus (EEEV), severeacute respiratory virus (SARS), Venezuelan equine encephalitis virus(VEEV), Vesicular stomatitis virus, Vibrio cholerae, serovar 01, Vibrioparahaemolyticus, West Nile virus, Wuchereria bancrofti, Yellow fevervirus, Yersinia enterocolitica, Yersinia pseudotuberculosis, andYersinia pestis.

Examples of infectious agents associated with human malignancies includeEpstein-Barr virus, Helicobacter pylori, Hepatitis B virus, Hepatitis Cvirus, Human heresvirus-8, Human immunodeficiency virus, Humanpapillomavirus, Human T cell leukemia virus, liver flukes, andSchistosoma haematobium.

A number of viruses are associated with viral hemorrhagic fever,including filoviruses (e.g., Ebola, Marburg, and Reston), arenaviruses(e.g. Lassa, Junin, and Machupo), and bunyaviruses. In addition,phleboviruses, including, for example, Rift Valley fever virus, havebeen identified as etiologic agents of viral hemorrhagic fever.Etiological agents of hemorrhagic fever and associated inflammation mayalso include paramyxoviruses, particularly respiratory syncytial virus(Feldmann, H. et al. (1993) Arch Virol Suppl. 7:81-100). In addition,other viruses causing hemorrhagic fevers in man have been identified asbelonging to the following virus groups: togavirus (Chikungunya),flavivirus (dengue, yellow fever, Kyasanur Forest disease, Omskhemorrhagic fever), nairovirus (Crimian-Congo hemorrhagic fever) andhantavirus (hemorrhagic fever with renal syndrome, nephropathicepidemia). Furthermore, Sin Nombre virus was identified as the etiologicagent of the 1993 outbreak of hantavirus pulmonary syndrome in theAmerican Southwest.

Target antigens may include proteins, or variants or fragments thereof,produced by any of the infectious organisms described herein, such as,but not limited to, viral coat proteins, i.e., influenza neuraminidaseand hemagglutinin, HIV gp160 or derivatives thereof, HIV Gag, HIV Nef,HIV Pol, SARS coat proteins, herpes virion proteins, WNV proteins, etc.Target antigens may also include bacterial surface proteins includingpneumococcal PsaA, PspA, LytA, surface or virulence associated proteinsof bacterial pathogens such as Nisseria gonnorhea, outer membraneproteins or surface proteases.

Target antigens may also include proteins, or variants or fragmentsthereof, of infectious agents associated with human malignancies such asthe human papillomavirus (HPV) oncoproteins E6 and E7. In certainembodiments, the oncoprotein may be modified to produce a non-oncogenicvariant or a variant having reduced oncogenicity relative to the wildtype protein. For example, the portion of the peptide that isresponsible for binding a tumor suppressor protein (e.g., p53 and pRb)may be deleted or modified so that it no longer interacts with the tumorsuppressor protein. As another example, an oncoprotein that is a kinase,such as Her2/neu, may be kinase-inactivated, e.g., by point mutation. Insome instances, two or more target antigens may be used duringimmunization. For example, the E6 and E7 antigens can be combined in afusion protein, or separate vectors containing heterologous nucleotidesencoding the modified or unmodified E6 and E7 target antigens are usedin combination. For example, an Ad5-E6 vector can be administered withan Ad5-E7 vector. In this example, the Ad5-E6 vector and Ad5-E7 vectormay be administered simultaneously or they may be administeredsequentially.

Target antigens of the present invention include but are not limited toantigens derived from a variety of tumor proteins. Illustrative tumorproteins useful in the present invention include, but are not limited toany one or more of, WT1, HPV E6, HPV E7, p53, MAGE-A1, MAGE-A2, MAGE-A3,MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6, -10, GAGE-1, -2, -8,GAGE-3, -4, -5, -6, -7B, NA88-A, NY-ESO-1, MART-1, MC1R, Gp100, PSA,PSM, Tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, Her2/neu,BRCA1, hTERT, hTRT, iCE, MUC1, MUC2, PRAME, P15, RU1, RU2, SART-1,SART-3, WT1, AFP, β-catenin/m, Caspase-8/m, CEA, CDK-4/m, ELF2M, GnT-V,G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin/m, RAGE,SART-2, TRP-2/INT2, 707-AP, Annexin II, CDC27/m, TPI/mbcr-abl, ETV6/AML,LDLR/FUT, Pml/RARα, and TEL/AML1. These and other tumor proteins areknown to the skilled artisan.

In certain embodiments tumor antigens may be identified directly from anindividual with cancer. In this regard, screens can be carried out usinga variety of known technologies. For example, in one embodiment, a tumorbiopsy is taken from a patient, RNA is isolated from the tumor cells andscreened using a gene chip (for example, from Affymetrix, Santa Clara,Calif.) and a tumor antigen is identified. Once the tumor target antigenis identified, it may then be cloned, expressed and purified usingtechniques known in the art. This target molecule is then linked to oneor more epitopes/cassettes of the present invention as described hereinand administered to the cancer patient in order to alter the immuneresponse to the target molecule isolated from the tumor. In this manner,“personalized vaccines” are contemplated within the context of theinvention. In certain embodiments, cancers may include carcinomas orsarcomas.

The adenovirus vectors of the present invention may also include nucleicacid sequences that encode proteins that increase the immunogenicity ofthe target antigen. In this regard, the protein produced followingimmunization with the adenovirus vector containing such a protein may bea fusion protein comprising the target antigen of interest fused to aprotein that increases the immunogenicity of the target antigen ofinterest.

In one embodiment, such an “immunological fusion partner” is derivedfrom a Mycobacterium sp., such as a Mycobacterium tuberculosis-derivedRa12 fragment. Ra12 compositions and methods for their use in enhancingthe expression and/or immunogenicity of heterologouspolynucleotide/polypeptide sequences are described in U.S. PatentApplication 60/158,585. Briefly, Ra12 refers to a polynucleotide regionthat is a subsequence of a Mycobacterium tuberculosis MTB32A nucleicacid. MTB32A is a serine protease of 32 KD molecular weight encoded by agene in virulent and avirulent strains of M. tuberculosis. Thenucleotide sequence and amino acid sequence of MTB32A have beendescribed (for example, U.S. Patent Application 60/158,585; see also,Skeiky et al., Infection and Immun. 67:3998-4007 (1999), incorporatedherein by reference). C-terminal fragments of the MTB32A coding sequenceexpress at high levels and remain as soluble polypeptides throughout thepurification process. Moreover, Ra12 may enhance the immunogenicity ofheterologous immunogenic polypeptides with which it is fused. One Ra12fusion polypeptide comprises a 14 KD C-terminal fragment correspondingto amino acid residues 192 to 323 of MTB32A. Other Ra12 polynucleotidesgenerally comprise at least about 15 consecutive nucleotides, at leastabout 30 nucleotides, at least about 60 nucleotides, at least about 100nucleotides, at least about 200 nucleotides, or at least about 300nucleotides that encode a portion of a Ra12 polypeptide. Ra12polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a Ra12 polypeptide or a portion thereof) or maycomprise a variant of such a sequence. Ra12 polynucleotide variants maycontain one or more substitutions, additions, deletions and/orinsertions such that the biological activity of the encoded fusionpolypeptide is not substantially diminished, relative to a fusionpolypeptide comprising a native Ra12 polypeptide. Variants preferablyexhibit at least about 70% identity, more preferably at least about 80%identity and most preferably at least about 90% identity to apolynucleotide sequence that encodes a native Ra12 polypeptide or aportion thereof.

Within another embodiment, an immunological fusion partner is derivedfrom protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain embodiments, the first 109 residues ofa Lipoprotein D fusion partner is included on the N-terminus to providethe polypeptide with additional exogenous T-cell epitopes and toincrease the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS1 (hemagglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292, 1986). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within anotherembodiment, a repeat portion of LYTA may be incorporated into a fusionpolypeptide. A repeat portion is found in the C-terminal region startingat residue 178. One particular repeat portion incorporates residues188-305.

Methods of Use

The adenovirus vectors of the present invention can be used in a numberof vaccine settings for generating an immune response against one ormore target antigens as described herein. The adenovirus vectors are ofparticular importance because of the unexpected finding that they can beused to generate immune responses in subjects who have preexistingimmunity to Ad and can be used in vaccination regimens that includemultiple rounds of immunization using the adenovirus vectors, regimensnot possible using previous generation adenovirus vectors.

Generally, generating an immune response comprises an induction of ahumoral response and/or a cell-mediated response. In certainembodiments, it is desirable to increase an immune response against atarget antigen of interest. In certain circumstances, generating animmune response may involve a decrease in the activity and/or number ofcertain cells of the immune system or a decrease in the level and/oractivity of certain cytokines or other effector molecules. As such“generating an immune response” or “inducing an immune response”comprises any statistically significant change, e.g. increase ordecrease, in the number of one or more immune cells (T cells, B cells,antigen-presenting cells, dendritic cells, neutrophils, and the like) orin the activity of one or more of these immune cells (CTL activity, HTLactivity, cytokine secretion, change in profile of cytokine secretion,etc.).

The skilled artisan would readily appreciate that a number of methodsfor establishing whether an alteration in the immune response has takenplace are available. A variety of methods for detecting alterations inan immune response (e.g. cell numbers, cytokine expression, cellactivity) are known in the art and are useful in the context of theinstant invention. Illustrative methods are described in CurrentProtocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek,David H. Margulies, Ethan M. Shevach, Warren Strober (2001 John Wiley &Sons, NY, N.Y.) Ausubel et al. (2001 Current Protocols in MolecularBiology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, N.Y.);Sambrook et al. (1989 Molecular Cloning, Second Ed., Cold Spring HarborLaboratory, Plainview, N.Y.); Maniatis et al. (1982 Molecular Cloning,Cold Spring Harbor Laboratory, Plainview, N.Y.) and elsewhere.Illustrative methods useful in this context include intracellularcytokine staining (ICS), ELISpot, proliferation assays, cytotoxic T cellassays including chromium release or equivalent assays, and geneexpression analysis using any number of polymerase chain reaction (PCR)or RT-PCR based assays.

In certain embodiments, generating an immune response comprises anincrease in target antigen-specific CTL activity of between 1.5 and 5fold in a subject administered the adenovirus vectors of the inventionas compared to a control. In another embodiment, generating an immuneresponse comprises an increase in target-specific CTL activity of about2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10,10.5, 11, 11.5, 12, 12.5, 15, 16, 17, 18, 19, 20, or more fold in asubject administered the adenovirus vectors as compared to a control.

In a further embodiment, generating an immune response comprises anincrease in target antigen-specific HTL activity, such as proliferationof helper T cells, of between 1.5 and 5 fold in a subject administeredthe adenovirus vectors of the invention that comprise nucleic acidencoding the target antigen as compared to an appropriate control. Inanother embodiment, generating an immune response comprises an increasein target-specific HTL activity of about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5,6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 15, 16,17, 18, 19, 20, or more fold as compared to a control. In this context,HTL activity may comprise an increase as described above, or decrease,in production of a particular cytokine, such as interferon-gamma(IFN-γ), interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-7, IL-12, IL-15,tumor necrosis factor-alpha (TNF-α), granulocyte macrophagecolony-stimulating factor (GM-CSF), granulocyte-colony stimulatingfactor (G-CSF), or other cytokine. In this regard, generating an immuneresponse may comprise a shift from a Th2 type response to a Th1 typeresponse or in certain embodiments a shift from a Th1 type response to aTh2 type response. In other embodiments, generating an immune responsemay comprise the stimulation of a predominantly Th1 or a Th2 typeresponse.

In a further embodiment, generating an immune response comprises anincrease in target-specific antibody production of between 1.5 and 5fold in a subject administered the adenovirus vectors of the presentinvention as compared to an appropriate control. In another embodiment,generating an immune response comprises an increase in target-specificantibody production of about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 15, 16, 17, 18, 19,20, or more fold in a subject administered the adenovirus vector ascompared to a control.

Thus the present invention provides methods for generating an immuneresponse against a target antigen of interest comprising administeringto the individual an adenovirus vector comprising: a) a replicationdefective adenovirus vector, wherein the adenovirus vector has adeletion in the E2b region, and b) a nucleic acid encoding the targetantigen; and readministering the adenovirus vector at least once to theindividual; thereby generating an immune response against the targetantigen. In certain embodiments, the present invention provides methodswherein the vector administered is not a gutted vector. In particularembodiments, the target antigen may be a wild-type protein, or afragment or variant thereof.

In a further embodiment, the present invention provides methods forgenerating an immune response against a target antigen in an individual,wherein the individual has preexisting immunity to Ad, by administeringto the individual an adenovirus vector comprising: a) a replicationdefective adenovirus vector, wherein the adenovirus vector has adeletion in the E2b region, and b) a nucleic acid a encoding the targetantigen; and readministering the adenovirus vector at least once to theindividual; thereby generating an immune response against the targetantigen. In particular embodiments, the target antigen may be awild-type protein, or a fragment or variant thereof.

With regard to preexisting immunity to Ad, this can be determined usingmethods known in the art, such as antibody-based assays to test for thepresence of Ad antibodies. Further, in certain embodiments, the methodsof the present invention include first determining that an individualhas preexisting immunity to Ad then administering the E2b deletedadenovirus vectors of the invention as described herein.

One embodiment of the invention provides a method of generating animmune response against one or more target antigens in an individualcomprising administering to the individual a first adenovirus vectorcomprising a replication defective adenovirus vector, wherein theadenovirus vector has a deletion in the E2b region, and a nucleic acidencoding at least one target antigen; administering to the individual asecond adenovirus vector comprising a replication defective adenovirusvector, wherein the adenovirus vector has a deletion in the E2b region,and a nucleic acid encoding at least one target antigen, wherein the atleast one target antigen of the second adenovirus vector is the same ordifferent from the at least one target antigen of the first adenovirusvector. In particular embodiments, the target antigen may be a wild-typeprotein, or a fragment or variant thereof.

Thus, the present invention contemplates multiple immunizations with thesame E2b deleted adenovirus vector or multiple immunizations withdifferent E2b deleted adenovirus vectors. In each case, the adenovirusvectors may comprise nucleic acid sequences that encode one or moretarget antigens as described elsewhere herein. In certain embodiments,the methods comprise multiple immunizations with an E2b deletedadenovirus encoding one target antigen, and re-administration of thesame adenovirus vector multiple times, thereby inducing an immuneresponse against the target antigen.

In a further embodiment, the methods comprise immunization with a firstadenovirus vector that encodes one or more target antigens, and thenadministration with a second adenovirus vector that encodes one or moretarget antigens that may be the same or different from those antigensencoded by the first adenovirus vector. In this regard, one of theencoded target antigens may be different or all of the encoded antigensmay be different, or some may be the same and some may be different.Further, in certain embodiments, the methods include administering thefirst adenovirus vector multiple times and administering the secondadenovirus multiple times. In this regard, the methods compriseadministering the first adenovirus vector 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, or more times and administering the secondadenovirus vector 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ormore times. The order of administration may comprise administering thefirst adenovirus one or multiple times in a row followed byadministering the second adenovirus vector one or multiple times in arow. In certain embodiments, the methods include alternatingadministration of the first and the second adenovirus vectors as oneadministration each, two administrations each, three administrationseach, and so on. In certain embodiments, the first and the secondadenovirus vectors are administered simultaneously. In otherembodiments, the first and the second adenovirus vectors areadministered sequentially.

As would be readily understood by the skilled artisan, more than twoadenovirus vectors may be used in the methods of the present invention.Three, 4, 5, 6, 7, 8, 9, 10 or more different adenovirus vectors may beused in the methods of the invention. In certain embodiments, themethods comprise administering more than one E2b deleted adenovirusvector at a time. In this regard, immune responses against multipletarget antigens of interest can be generated by administering multipledifferent adenovirus vectors simultaneously, each comprising nucleicacid sequences encoding one or more antigens.

The present invention provides methods of generating an immune responseagainst any target antigen, such as those described elsewhere herein.

The present invention provides methods of generating an immune responseagainst any infectious agent, such as those described elsewhere herein.

As noted elsewhere herein, the adenovirus vectors of the inventioncomprise nucleic acid sequences that encode one or more target antigensof interest from any one or more of the infectious agents against whichan immune response is to be generated. For example, target antigens mayinclude, but are not limited to, viral coat proteins, i.e., influenzaneuraminidase and hemagglutinin, HIV gp160, p24, gp120, gp41, envelope,protease, or reverse transcriptase, or derivatives of any of these viralproteins; SARS coat proteins, herpes virion proteins, WNV proteins, etc.Target antigens may also include bacterial surface proteins includingpneumococcal PsaA, PspA, LytA, surface or virulence associated proteinsof bacterial pathogens such as Nisseria gonnorhea, outer membraneproteins or surface proteases. In particular embodiments, the targetantigen may be a wild-type protein, or a fragment or variant thereof.

In certain embodiments, the adenovirus vectors are used to generate animmune response against a cancer. In this regard, the methods includegenerating an immune response against carcinomas or sarcomas such assolid tumors, lymphomas or leukemias. Thus, the adenovirus vectorsdescribed herein are used to generate an immune response against acancer including but not limited to carcinomas or sarcomas such asneurologic cancers, melanoma, non-Hodgkin's lymphoma, Hodgkin's disease,leukemias, plasmocytomas, adenomas, gliomas, thymomas, breast cancer,prostate cancer, colo-rectal cancer, kidney cancer, renal cellcarcinoma, uterine cancer, pancreatic cancer, esophageal cancer, lungcancer, ovarian cancer, cervical cancer, testicular cancer, gastriccancer, multiple myeloma, hepatoma, acute lymphoblastic leukemia (ALL),acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML),and chronic lymphocytic leukemia (CLL), or other cancers.

Further, in this regard, the cancer target antigens may include but arenot limited to antigens derived from a variety of tumor proteins.Illustrative tumor proteins useful in the present invention include, butare not limited to any one or more of, p53, HPV E6, HPV E7, MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6,-10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, NY-ESO-1, MART-1,MC1R, Gp100, PSA, PSM, Tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA,Cyp-B, Her2/neu, hTERT, hTRT, iCE, MUC1, MUC2, PRAME, P15, RU1, RU2,SART-1, SART-3, WT1, AFP, β-catenin/m, Caspase-8/m, CEA, CDK-4/m, ELF2M,GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin/m,RAGE, SART-2, TRP-2/INT2, 707-AP, Annexin II, CDC27/m, TPI/mbcr-abl,ETV6/AML, LDLR/FUT, Pml/RARα, and TEL/AML1. These and other tumorproteins are known to the skilled artisan.

Methods are also provided for treating or ameliorating the symptoms ofany of the infectious diseases or cancers as described herein. Themethods of treatment comprise administering the adenovirus vectors oneor more times to individuals suffering from or at risk from sufferingfrom an infectious disease or cancer as described herein. As such, thepresent invention provides methods for vaccinating against infectiousdiseases or cancers in individuals who are at risk of developing such adisease. Individuals at risk may be individuals who may be exposed to aninfectious agent at some time or have been previously exposed but do notyet have symptoms of infection or individuals having a geneticpredisposition to developing a cancer or being particularly susceptibleto an infectious agent.

The present invention contemplates the use of adenovirus vectors for thein vivo delivery of nucleic acids encoding a target antigen, or afragment or variant thereof. Once injected into a subject, the nucleicacid sequence is expressed resulting in an immune response against theantigen coded for by the sequence. The adenovirus vector vaccine isadministered in an “effective amount”, that is, an amount of adenovirusvector that is effective in a selected route or routes of administrationto elicit an immune response as described elsewhere herein. In certainembodiments, an effective amount is one that induces an immune responseeffective to facilitate protection or treatment of the host against thetarget infectious agent or cancer. The amount of vector in each vaccinedose is selected as an amount which induces an immune, immunoprotectiveor other immunotherapeutic response without significant adverse effectsgenerally associated with typical vaccines. Once vaccinated, subjectsmay be monitored to determine the efficacy of the vaccine treatment.Monitoring the efficacy of vaccination may be performed by any methodknown to a person of ordinary skill in the art. In some embodiments,blood or fluid samples may be assayed to detect levels of antibodies. Inother embodiments, ELISpot assays may be performed to detect acell-mediated immune response from circulating blood cells or fromlymphoid tissue cells.

The adenovirus vectors of the invention are generally prepared as knownin the art (see e.g., Hodges et al., 2000 supra; or Amalfitano et al.,1998 supra). For example, in certain embodiments, tissue culture platescontaining E.C7 or C-7 cells are infected with the adenovirus vectorvirus stocks at an appropriate MOI (e.g., 5) and incubated at 37.0° C.for 40 h. The infected cells are harvested, resuspended in anappropriate buffer such as 10 mM Tris-CI (pH 8.0), and sonicated, andthe virus is purified by two rounds of cesium chloride densitycentrifugation. In certain techniques, the virus containing band isdesalted over a Sephadex CL-6B column (Pharmacia Biotech, Piscataway,N.J.), glycerol is added to a concentration of 12%, and aliquots arestored at −80° C. The titer of the stock is measured (e.g., bymeasurement of the optical density at 260 nm of an aliquot of the virusafter SDS lysis). GMP procedures for producing appropriate Ad stocks forhuman administration are used where appropriate.

For administration, the adenovirus vector stock is combined with anappropriate buffer, physiologically acceptable carrier, excipient or thelike. In certain embodiments, an appropriate number of adenovirus vectorparticles are administered in an appropriate buffer, such as, sterilePBS. In certain circumstances it will be desirable to deliver theadenovirus vector compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally. In certainembodiments, solutions of the active compounds as free base orpharmacologically acceptable salts may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. In other embodiments, E2b deleted adenovirusvectors may be delivered in pill form, delivered by swallowing or bysuppository.

Illustrative pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (for example, see U.S. Pat. No. 5,466,468). In all cases theform must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria, molds and fungi. The carriercan be a solvent or dispersion medium containing, for example, water,ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), suitable mixtures thereof, and/orvegetable oils. Proper fluidity may be maintained, for example, by theuse of a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and/or by the use ofsurfactants. The prevention of the action of microorganisms can befacilitated by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.In many cases, it will be preferable to include isotonic agents, forexample, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

In one embodiment, for parenteral administration in an aqueous solution,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. Moreover, for human administration, preparationswill of course preferably meet sterility, pyrogenicity, and the generalsafety and purity standards as required by FDA Office of Biologicsstandards.

The carriers can further comprise any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. The phrase “pharmaceutically-acceptable” refersto molecular entities and compositions that do not produce an allergicor similar untoward reaction when administered to a human.

In certain embodiments, the adenovirus vectors of the invention may beadministered in conjunction with one or more immunostimulants, such asan adjuvant. An immunostimulant refers to essentially any substance thatenhances or potentiates an immune response (antibody and/orcell-mediated) to an antigen. One type of immunostimulant comprises anadjuvant. Many adjuvants contain a substance designed to protect theantigen from rapid catabolism, such as aluminum hydroxide or mineraloil, and a stimulator of immune responses, such as lipid A, Bortadellapertussis or Mycobacterium tuberculosis derived proteins. Certainadjuvants are commercially available as, for example, Freund'sIncomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2(SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminumhydroxide gel (alum) or aluminum phosphate; salts of calcium, iron orzinc; an insoluble suspension of acylated tyrosine; acylated sugars;cationically or anionically derivatized polysaccharides;polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A andquil A. Cytokines, such as GM-CSF, interleukin-2, -7, -12, and otherlike growth factors, may also be used as adjuvants.

Within certain embodiments of the invention, the adjuvant composition ispreferably one that induces an immune response predominantly of the Th1type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within certain embodiments, in which a response ispredominantly Th1-type, the level of Th1-type cytokines will increase toa greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, Ann. Rev.Immunol. 7:145-173, 1989.

Certain illustrative adjuvants for eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A, together with analuminum salt. MPL® adjuvants are available from GlaxoSmithKlein(Research Triangle Park, N.C.; see, for example, U.S. Pat. Nos.4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containingoligonucleotides (in which the CpG dinucleotide is unmethylated) alsoinduce a predominantly Th1 response. Such oligonucleotides are wellknown and are described, for example, in WO 96/02555, WO 99/33488 andU.S. Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequencesare also described, for example, by Sato et al., Science 273:352, 1996.Another adjuvant for use in the present invention comprises a saponin,such as Quil A, or derivatives thereof, including QS21 and QS7 (AquilaBiopharmaceuticals Inc., Framingham, Mass.); Escin; Digitonin; orGypsophila or Chenopodium quinoa saponins. Other formulations mayinclude more than one saponin in the adjuvant combinations of thepresent invention, for example combinations of at least two of thefollowing group comprising QS21, QS7, Quil A, β-escin, or digitonin.

In certain embodiments, the compositions may be delivered by intranasalsprays, inhalation, and/or other aerosol delivery vehicles. The deliveryof drugs using intranasal microparticle resins (Takenaga et al., JControlled Release 1998 Mar. 2; 52(1-2):81-7) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) arewell-known in the pharmaceutical arts. Likewise, illustrativetransmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045.

In certain embodiments, liposomes, nanocapsules, microparticles, lipidparticles, vesicles, and the like, are used for the introduction of thecompositions of the present invention into suitable hostcells/organisms. In particular, the compositions of the presentinvention may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike. Alternatively, compositions of the present invention can be bound,either covalently or non-covalently, to the surface of such carriervehicles.

The formation and use of liposome and liposome-like preparations aspotential drug carriers is generally known to those of skill in the art(see for example, Lasic, Trends Biotechnol 1998 July; 16(7):307-21;Takakura, Nippon Rinsho 1998 March; 56(3):691-5; Chandran et al., IndianJ Exp Biol. 1997 August; 35(8):801-9; Margalit, Crit Rev Ther DrugCarrier Syst. 1995; 12(2-3):233-61; U.S. Pat. Nos. 5,567,434; 5,552,157;5,565,213; 5,738,868 and 5,795,587, each specifically incorporatedherein by reference in its entirety).

Liposomes have been used successfully with a number of cell types thatare normally difficult to transfect by other procedures, including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., J Biol Chem. 1990 Sep. 25; 265(27):16337-42; Muller et al., DNACell Biol. 1990 April; 9(3):221-9). Liposomes have been used effectivelyto introduce genes, various drugs, radiotherapeutic agents, enzymes,viruses, transcription factors, allosteric effectors and the like, intoa variety of cultured cell lines and animals. Furthermore, the use ofliposomes does not appear to be associated with autoimmune responses orunacceptable toxicity after systemic delivery.

In certain embodiments, liposomes are formed from phospholipids that aredispersed in an aqueous medium and spontaneously form multilamellarconcentric bilayer vesicles (also termed multilamellar vesicles (MLVs).

Alternatively, in other embodiments, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (see, for example, Quintanar-Guerrero etal., Drug Dev Ind Pharm. 1998 December; 24(12):1113-28). To avoid sideeffects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 μm) may be designed using polymers able tobe degraded in vivo. Such particles can be made as described, forexample, by Couvreur et al., Crit Rev Ther Drug Carrier Syst. 1988;5(1):1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998 March;45(2):149-55; Zambaux et al. J Controlled Release. 1998 Jan. 2;50(1-3):31-40; and U.S. Pat. No. 5,145,684.

Routes and frequency of administration of the therapeutic compositionsdescribed herein, as well as dosage, will vary from individual toindividual, and from disease to disease, and may be readily establishedusing standard techniques. In general, the pharmaceutical compositionsand vaccines may be administered by injection (e.g., intracutaneous,intramuscular, intravenous or subcutaneous), intranasally (e.g., byaspiration), in pill form (e.g. swallowing, suppository for vaginal orrectal delivery). In certain embodiments, between 1 and 10 doses may beadministered over a 52 week period. In certain embodiments, 6 doses areadministered, at intervals of 1 month, and further booster vaccinationsmay be given periodically thereafter. Alternate protocols may beappropriate for individual patients. As such, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more doses may beadministered over a 1 year period or over shorter or longer periods,such as over 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100week periods. Doses may be administered at 1, 2, 3, 4, 5, or 6 weekintervals or longer intervals.

A suitable dose is an amount of an adenovirus vector that, whenadministered as described above, is capable of promoting a targetantigen immune response as described elsewhere herein. In certainembodiments, the immune response is at least 10-50% above the basal(i.e., untreated) level. Such response can be monitored by measuring thetarget antigen(s) antibodies in a patient or by vaccine-dependentgeneration of cytolytic effector cells capable of killing patient tumoror infected cells in vitro, or other methods known in the art formonitoring immune responses. Such vaccines should also be capable ofcausing an immune response that leads to an improved clinical outcome ofthe disease in question in vaccinated patients as compared tonon-vaccinated patients.

In general, an appropriate dosage and treatment regimen provides theadenovirus vectors in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome for the particular disease being treated intreated patients as compared to non-treated patients. Such improvementsin clinical outcome would be readily recognized by a treating physician.Increases in preexisting immune responses to a target protein generallycorrelate with an improved clinical outcome. Such immune responses maygenerally be evaluated using standard proliferation, cytotoxicity orcytokine assays, which may be performed using samples obtained from apatient before and after treatment.

While one advantage of the present invention is the capability toadminister multiple vaccinations with the same or different adenovirusvectors, particularly in individuals with preexisting immunity to Ad,the adenoviral vaccines of this invention may also be administered aspart of a prime and boost regimen. A mixed modality priming and boosterinoculation scheme may result in an enhanced immune response. Thus, oneaspect of this invention is a method of priming a subject with a plasmidvaccine, such as a plasmid vector comprising a target antigen ofinterest, by administering the plasmid vaccine at least one time,allowing a predetermined length of time to pass, and then boosting byadministering the adenovirus vector. Multiple primings, e.g., 1-4, maybe employed, although more may be used. The length of time betweenpriming and boost may typically vary from about four months to a year,but other time frames may be used. In certain embodiments, subjects maybe primed four times with plasmid vaccines, and then boosted 4 monthslater with the adenovirus vector.

EXAMPLES Example 1 Multiple Injections of Ad5Null Adenovirus VectorProduces Anti-Adenovirus Antibodies

This example shows that multiple injections of Ad5-null results in theproduction of anti-adenovirus antibodies in the injected subjects

It was demonstrated that the Ad5Null adenovirus vector that does notcontain any heterologous nucleic acid sequences, generates aneutralizing immune response in mice. In particular, in one experiment,female BALB/c mice aged 5-7 weeks were immunized with Ad5Null viralparticles at 14 day intervals. To determine the presence ofanti-adenovirus antibodies, an enzyme linked immunosorbent assay (ELISA)was used. For this ELISA, 10⁹ viral particles were coated ontomicrotiter wells in 100 μL of 0.05M carbonate/bicarbonate buffer, pH9.6, and incubated overnight at room temperature. For a standardimmunoglobulin G (IgG) reference curve, 200 ng, 100 ng, 50 ng, 25 ng,and 0 ng of purified mouse IgG (Sigma Chemicals) were coated ontomicrotiter wells as described above. After incubation, all wells werewashed 3 times with 250 μL of 1% bovine serum albumin (BSA) in phosphatebuffered saline (PBS), pH 7.4. After washing, 250 μL of BSA/PBS wasadded to all and incubated for 30 minutes at room temperature to blockunbound sites. After incubation, all wells were washed 3 times with 250μL of BSA/PBS. After washing, 200 μL of a 1/100 serum dilution inBSA/PBS was added to wells and incubated for 1 hour at room temperature.For a positive control, 200 μL of a 1/10000 dilution of anti-adenovirusantiserum (Biodesign International) in BSA/PBS were added to wells.Control wells contained BSA/PBS only. After incubation, all wells werewashed 3 times with 250 μL of BSA/PBS. After washing, 200 μL of a1/10000 dilution of peroxidase conjugated gamma chain specific goatanti-mouse IgG (Sigma Chemicals) in BSA/PBS were added to each well andincubated for 1 hour at room temperature. After incubation, all wellswere washed 3 times with 250 μL of BSA/PBS. After washing, 200 μL ofdeveloping reagent (0.5 mg/mL 1,2 phenylenediamine in 0.2M potassiumphosphate buffer, pH 5.0, containing 0.06% hydrogen peroxide) was addedto each well and incubated for 30-40 minutes at room temperature. Afterincubation, the color reaction was stopped by addition of 50 μL 5N HClto each well. All wells were then read in a microwell plate reader at492 nm. After readings were obtained, the optical density readings ofunknown samples were correlated with the standard IgG curve to obtainthe nanograms of IgG bound per well. This was performed using the INSTATstatistical package.

As shown in FIG. 1, significant levels (P<0.001) of anti-adenovirus IgGantibody were detected in mice 2 weeks after a first injection with 10¹⁰Ad-5-null. A significantly higher level (P<0.001) was observed 2 weeksafter a second injection with 10¹⁰ adenovirus. Significantly higher(P<0.001) levels of antibody were continued to be observed 2 weeks aftera third injection with 10¹⁰ Ad5-null. Each value represents the averageof triplicate determinations from pooled sera of 5 mice in each group.These results indicate that multiple injections of Ad5-null results inthe production of anti-adenovirus antibodies in the injected subjects.

To determine the presence of neutralizing antibody to Ad, the followingassay was utilized. A HEK-293T cell line was cultured in 200 μL ofculture medium consisting of DMEM containing 10% fetal calf serum(DMEM/FCS) in microwell tissue culture plates at a cell concentration of2×10³ cells per well for 24 hours at 37 C in 5% CO₂. After incubation,100 μL of culture medium was removed from triplicate wells and mixedwith 20 μL of DMEM/FCS containing viral particles (VP). After mixing,the 120 μL mixture was added back to the respective microwells. Inanother set of triplicate wells, 100 μL of culture medium was removedand mixed with 20 μL of heat inactivated (56 C for 1 hour) Ad immunemouse serum previously incubated with VP for one hour at roomtemperature.

After mixing, the 120 μL mixture was added back to the respective wells.In triplicate cell control wells, 20 μL of DMEM/FCS was added to controlfor total culture medium volume. Triplicate medium only control wellscontained 220 μL of DMEM/FCS. The tissue culture plate was incubated foran additional 3 days at 37 C in 5% CO₂. After incubation, 40 μL ofPROMEGA cell viability reagent (Owen's reagent) was added to all wellsand incubated for 75 minutes at 37 C in 5% CO₂. In this assay, theOwen's reagent (MTS tetrazolium compound) is bioreduced by viable cellsinto a colored formazan product that is soluble in tissue culturemedium. The quantity of formazan product as measured by absorbance at490 nm is directly proportional to the number of living cells inculture. After incubation, 150 μL was removed from each well andtransferred to another microwell plate for optical density readings.Optical density readings at 492 nm were subsequently obtained using amicrowell plate reader.

In an experiment to detect the presence of neutralizing antibodies toAd, groups of 5 mice each were injected once, twice, or three times with10¹⁰ Ad5null at two week intervals. Two weeks after the final injectionof virus, mice were bled, pooled, and assessed for neutralizing antibodyas described above using 4×10⁷ VP incubated with or without heatinactivated sera. Cells cultured alone served as a control group. Asshown in FIG. 2, normal mice and mice injected one time with Ad5null didnot exhibit significant levels of neutralizing antibody. Mice injectedtwo times with Ad exhibited significant (P<0.05) levels of neutralizingantibody as compared with cells incubated with virus only. Mice injectedthree times with Ad5-null also exhibited significant (P<0.01) levels ofneutralizing antibody as compared with cells incubated with virus only.

Example 2 Multiple Injections of an E2B Deleted Adenovirus VectorGenerates an Immune Response Against Target Antigens

This example shows that multiple injections of an E2b deleted adenovirusvector containing HIV-gag results in the production of HIV-gag immunity.

Two groups of mice were used for this experiment. One group served as anormal control group. The second group was injected 3 times with E2bdeleted Ad containing HIV-gag at 2 week intervals. Four weeks after thelast injection, mice were bled and assessed for IgG antibody levelsusing an ELISA assay as follows: For this assay, 100 ng of a purifiedmixture of HIV-gag proteins p17/p24 were coated onto microtiter wells in100 μL of 0.05M carbonate/bicarbonate buffer, pH 9.6, and incubatedovernight at room temperature. For a standard IgG reference curve,purified mouse IgG (SigmaChemicals) in quantities of 200 ng, 100 ng, 50ng, 25 ng, and 0 ng were coated onto microtiter wells as describedabove. After incubation, all wells were washed 3 times with 250 μL of 1%bovine serum albumin (BSA) in phosphate buffered saline (PBS), pH 7.4.After washing, 200 μL of BSA/PBS was added to wells and incubated for 30minutes at room temperature to block any remaining sites in themicrotiter wells. After incubation, all wells were washed 3 times with250 μL of BSA/PBS. After washing, 200 μL of a 1/100 dilution of mouseserum in BSA/PBS was added to wells and incubated for one hour at roomtemperature. For a positive control, 100 ng of mouse monoclonal anti-p24IgG antibody in BSA/PBS was added to wells. Blank control wellscontained BSA/PBS only. After incubation, all wells were washed 3 timeswith 250 μL of BSA/PBS. After washing, 200 μL of a 1/10000 dilution ofperoxidase conjugated gamma chain specific goat anti-mouse IgG (SigmaChemicals) in BSA/PBS was added to each well and incubated for one hourat room temperature. After incubation, all wells were washed 3 timeswith 2504 of BSA/PBS. After washing, 200 μL of developing reagent (0.5mg/mL 1,2 phenylenediamine in 0.2M potassium phosphate buffer, pH 5.0,containing 0.06% hydrogen peroxide) was added to each well and incubatedfor 30-40 minutes at room temperature. After incubation, the colorreaction was stopped by addition of 50 μL 5M HCl to each well. All wellswere read in a microwell plate reader at 492 nm. After readings wereobtained, the optical density readings of unknown samples werecorrelated with the standard IgG curve to obtain nanograms of IgG boundper well. This was performed utilizing the INSTAT statistical package.

As shown in FIG. 3, low but significant levels of Gag IgG were detectedin mice 2 weeks after a second injection and 4 weeks after a thirdinjection with 10¹⁰ E2b deleted Ad containing the HIV-gag gene.Moreover, when compared with respective pre-injections bleeds, low butsignificant levels (P<0.01) of detectable antibody were observed up to 8weeks (Day 84) as well as 13 weeks (Day 119) post 3^(rd) injection withE2b deleted Ad-gag vector vaccine.

Example 3 Multiple Injections of an E2B Deleted Adenovirus VectorGenerates an Immune Response Against Multiple Target Antigens

This example demonstrates that mice injected multiple times with an E2bdeleted adenovirus vector vaccine expressing a first target antigen(HIV-gag) and subsequently injected multiple times with an E2b deletedadenovirus vector expressing a second target antigen (β-galactosidase)produce an immune response against the first and the second antigen.

A group of five mice were injected 3 times at 2 week intervals with 10¹⁰E2b deleted adenovirus vector vaccine containing the HIV-gag gene. Fourweeks later, the mice were injected two times at a weekly interval with10¹⁰ E2b deleted adenovirus vector vaccine containing β-galactosidase. Agroup of 5 mice served as a normal control group. Sera from miceinjected only with E2b deleted Ad-βgal that showed high levels ofβ-galactosidase IgG antibody served as a positive control.

To determine the presence of β-galactosidase antibodies, an enzymelinked immunosorbent assay (ELISA) was used. For this ELISA, 100 ng ofpurified β-galactosidase was coated onto microtiter wells in 100 μL of0.05M carbonate/bicarbonate buffer, pH 9.6 and incubated over night atroom temperature. For a standard IgG reference curve, purified mouse IgG(Sigma Chemicals) in quantities of 200 ng, 100 ng, 50 ng, 25 ng, and 0ng were coated onto microtiter wells as described above. Afterincubation, all wells were washed 3 times with 250 μL of 1% bovine serumalbumin (BSA) in phosphate buffered saline (PBS), pH 7.4. After washing,250 μL of BSA/PBS was added to all wells and incubated for 30 minutes atroom temperature. After incubation, all were washed 3 times with 250 μLof BSA/PBS. After washing, 200 μL of a 1/100 of serum in BSA/PBS wasadded to wells and incubated for 1 hour at room temperature. Blankcontrol wells contained BSA/PBS only. After incubation, all wells werewashed 3 times with 250 μL of BSA/PBS. After washing, 200 μL of a1/10000 dilution of peroxidase conjugated gamma chain specific goatanti-mouse IgG (Sigma Chemicals) in BSA/PBS was added to each well andincubated for 1 hour at room temperature. After incubation, all wellswere washed 3 times with 250 μL of BSA/PBS. After washing, 200 μL ofdeveloping reagent (0.5 mg/mL 1, 2 phenylenediamine in 0.2M potassiumphosphate buffer, pH 5.0, containing 0.06% hydrogen peroxide) was addedto each well and incubated for 30-40 minutes at room temperature. Afterincubation, the color reaction was stopped by addition of 50 μL 5N HClto each well. All wells were then read in a microwell plate reader at492 nm. After readings were obtained, the optical density readings ofunknown samples were correlated with the standard IgG curve to obtainthe nanograms of IgG bound per well. This was performed utilizing theINSTAT statistical package.

As shown in FIG. 4, sera assessed 2 weeks after the last injection frommice injected with E2b deleted adenovirus vector containing E. coliβ-galactosidase exhibited significantly (P<0.01) higher levels ofanti-β-galactosidase IgG antibodies as compared to normal control mice.Detectable levels of anti-β-galactosidase antibody persisted up to 5weeks after the last injection. Moreover, significant levels of HIV-gagimmunity were still readily detected and levels of these antibodies evenincreased.

In a related experiment, cellular immune responses in mice were assessedafter multiple immunizations. Mice were immunized with E2b deletedadenovirus vector vaccine containing the HIV-gag gene three times at 14day intervals. Four weeks later, the mice were subsequently immunizedwith E2b deleted adenovirus vector vaccine containing theβ-galactosidase gene twice at 14 day intervals. ELISpot assays wereperformed to determine cellular mediated immune responses in vaccinatedsubjects. ELISpot assay kits were obtained from eBioscience and assayplates were prepared according to manufacturer's specifications. Theassay was performed as described in the manufacturer's instructions.Briefly, capture antibody was coated onto ELISpot assay plates perinstructions and coated overnight. After washing and blocking unboundsites, mitogen, specific antigen, and controls were added to wells incomplete RPMI-1640 culture medium at 100 μL per well. Spleen cells frommice were harvested and prepared for cell culture. Cells were then addedto wells in a 100 μL volume at the desired cell density. The ELISpotplates were then incubated at 37 C in a 5% CO₂ humidified incubator forapproximately 48 hours. After incubation, the ELISpot plates weredeveloped according to manufacturer's instructions. The data wasexpressed as the number of spot forming cells (SPC) per 10⁶ splenocytes.

As shown in FIG. 5A, ELISpot analysis showed that mice exhibited theproduction interferon-γ (IFN-γ) upon re-stimulation to HIV-gag andAd5null virions. Furthermore, as shown in FIG. 5B, ELISpot analysis alsoshowed the production of interleukin-2 (IL-2) upon re-stimulation withHIV-gag, β-galactosidase, and Ad5null virions. These results indicatedthat cell-mediated immune responses could be generated in the samesubjects after multiple injections with E2b deleted adenovirus vectorvaccines encoding 2 differing target antigens. Moreover, thecell-mediated immune response against the second immunizing antigenoccurred in the presence of immunity to Ad.

Thus, the E2b deleted adenovirus vectors of the present invention can beused to immunize against multiple antigens using regimens of multipleimmunizations.

Example 4 Cell Mediated Immune (CMI) Response Induction in Adenovirus 5Immune Cynomolgus Macaques

This example shows that multiple injections of an E2b deleted adenovirusvector containing HIV-gag results in the production of HIV-gag immunity,even in the presence of Ad5 immunity.

Three non-human primates (NHP) were injected with a single dose of 10¹⁰VP viable wild type Ad5. Ad5 neutralizing antibody (NAb) was measured 30days after administration, and the NHP titers were ≥1:50 (FIG. 6). TheAd5 immune NHP were then immunized three times; days 0, 27, and 58; withAd5 [E1-, E2b-]-gag (10¹⁰ VP/dose). Peripheral blood mononuclear cells(PBMC) from individual NHP were collected at the indicated time points.CMI responses upon re-stimulation with HIV-gag protein were assayed 32days after the final immunization with Ad5 [E1-, E2b-]-gag. ELISpotanalysis indicated that PBMC from all three NHP responded similarly uponre-stimulation with HIV-gag protein with an average frequency of 223SFC/10⁶ PBMC producing INF-γ and 207 SFC/10⁶ PBMC producing IL-2 (FIG.7A and FIG. 7B, respectively). These values were significantly (P<0.05)elevated when compared to their baseline values. The Ad5 viral NAbtiters ranged from 1/1000 to 1/20,000 at the termination of this study(FIG. 6).

As evidenced by the induction of specific CMI responses to the HIV-gagprotein, these results indicate that NHP can be successfully immunizedwith the Ad5 [E1-, E2b-] vector platform in the presence of Adenovirus 5immunity.

Example 5 A Modified HER2 Cancer Vaccine Vector Based on the Ad5 [E1-,E2B-] Platform

This example shows that multiple immunizations of Ad5 immune mice withAd5 [E1-, E2b-]-HER2 induced HER2 specific cell mediated immuneresponses and antibody responses that had beneficial effects on tumorprogression.

Production and Characterization of Ad5 HER2 Vector

The Ad5 [E1-, E2b-]-HER2 vector was constructed by recombination inBJ5183 bacterial cells and rescued by transfection into pre-terminalprotein (pTP) and polymerase (pol) expressing E.C7 cells. The Ad5 [E1-,E2b-]-HER2 vector contains deletions in both the pol and pTP genes,since this arrangement has been demonstrated to result in decreasedliver toxicity when compared to Ad5 [E1-]. The mini-gene cassetteexpressing HER2 was subcloned into the E1 region of a shuttle vector andrecombined with E2b deleted (pol-, pTP-) Ad5 genomic DNA. The recombinedAd5 [E1-, E2b-]-HER2 was verified by restriction analysis and rescued asviruses by restriction enzyme release and transfection into E.C7 cells.

The HER2 gene utilized for these studies was a modified kinase-inactiveHER2 having a K753A mutation that renders it non-oncogenic (Morse, M. A.et al., Int. J. Cancer, 2009, Vol. 9999, Issue 999A, page NA, publishedonline 23 Oct. 2009 and Akiyama, T. et al., Mol. Cell. Biol. 1991,11(2):833-842).

The Ad5 [E1-, E2b-]-HER2 viral vector was manufactured by releasing fromE.C7 producer cells by Triton X-100, precipitated in PEG, purified onCsCl gradients, dialyzed against 20 mM HEPES (pH 7.4) containing 5%sucrose, aliquoted, and frozen in a dry ice-ethanol bath. Particleconcentration was calculated both by absorption at 260 and 280 nm(normal ratio=1.3). The infectivity of the virus particles (VP) wasmeasured in a plaque assay and the infectious units per total virusparticles (IU/VP) was 1/90.

Studies were performed to confirm HER2 gene expression of both Ad5 [E1-,E2b-]-HER2 vector platform. It was first determined that the HER2antigen could be expressed on cells transfected with the vectorplatform. A549 cells were obtained from ATCC and transfected with Ad5[E1-, E2b-]-HER2. Western Blot analysis was performed to determineHER2/neu expression by the Ad5 [E1-, E2b-]-HER2/neu vector platform.Briefly, 10⁶ human lung carcinoma cells (A-549) (ATCC number CCL-185)were infected at a multiplicity of infection (MOI) of 900 and 150 VP,incubated for 24 hr and then lysed. Cell lysates were separated on a 10%SDS-polyacrylamide gel and transferred onto a PVDF membrane (GEHealthcare, Piscataway, N.J.). The membranes were then blocked with TBScontaining 5% (w/v) blocking reagent (GE Healthcare, Piscataway, N.J.)for 2 hours at room temperature and sequentially incubated with mouseanti-HER2 antibody (1:250) (Genway, San Diego, Calif.) and goatanti-mouse-HRP conjugated antibody (1:1000) (Kirkegaard & PerryLaboratories, Gaithersburg, Md.) for one hour at room temperature.Reactivity was determined by chemilluminescence using an ECL WesternBlotting analysis system (GE Healthcare, Piscataway, N.J.) according tothe manufacturer's specifications. As shown in FIG. 8, a single HER2expressing band was observed.

Induction of Ad5 Immunity in Mice as Evidenced by Levels of Ad5Neutralizing Antibody

To induce Ad5 immunity, Ad5 naïve BALB/c mice were injectedintradermally twice at two week intervals with 10¹⁰ virus particles ofAd5-null (empty). Two weeks later, serum samples were collected andassessed for endpoint Ad5 neutralizing antibody (NAb) titers. As shownin FIG. 9, this protocol induced NAb activity in BALB/c mice. BALB/cmice were used because the HER2 expressing tumor line used for theimmunotherapy studies is implanted in this strain of mouse. In thestudies below, mice were made Ad5 immune by two injections withAd5-null.

Immunization of Ad5 Immune Mice with Ad5 [E1-, E2b-]-HER2

Experiments were performed to assess immune responses in Ad5 immune miceduring multiple immunizations with Ad5 vector based vaccines. Two weeksfollowing the last Ad5-null immunization, groups of female BALB/c mice,4 to 8 weeks old, were immunized one, two or three times at weeklyintervals with 10¹⁰ virus particles of Ad5 [E1-, E2b-]-HER2. Assessmentof sera after two injections with Ad5-null revealed that the Ad5 NAbendpoint NAb titers were 1/100. Thus, before mice were immunized, theirpre-existing Ad5 NAb endpoint titers were 1/100 (FIG. 10). Two weeksfollowing the last immunization with Ad5 [E1-, E2b-]-HER2, mice wereeuthanized and their spleens were harvested for analysis of CMI andantibody responses.

Induction of CMI Responses

Cell mediated immune responses were assessed by ELISpot assays performedon splenocytes exposed to HER2 peptides. Splenocytes from Ad5 immuneBALB/c mice that were immunized subcutaneously with Ad5 [E1-, E2b-]-HER2were harvested and assessed for the number of IFN-γ and IL-2 secretingsplenocytes. As shown in FIG. 11, elevated numbers of IFN-γ and IL-2secreting cells were observed in spleens assayed from mice after twoimmunizations with Ad5 [E1-, E2b-]-HER2. Moreover, the highest numbersof IFN-γ and IL-2 secreting cells were observed after the thirdimmunization.

Specificity studies revealed that immunizations induced specific HER2associated CMI responses and not responses against other irrelevantantigens such as the cytomegalovirus (CMV) antigen or β-galactosidase.As shown in FIG. 10, the HER2 CMI responses occurred despite thepresence of high levels (1/500 titer) of Ad5 NAb activity in seraresulting from multiple immunizations. These results demonstrate thatmultiple immunizations of Ad5 immune mice with Ad5 [E1-, E2b-]-HER2induced HER2 specific CMI responses.

Induction of Humoral Responses

Studies were also performed to determine if anti-HER2 IgG antibody wasinduced after immunizations. An ELISA for circulating IgG antibody toHER2 was performed. As shown in FIG. 12, increasing quantities ofdetectable antibody to HER2 were observed after one, two, and threeimmunizations with Ad5 [E1-, E2b-]-HER2, with the greatest quantities ofantibody observed after the third immunization. Statistical t testanalyses revealed that antibody levels were significantly (P<0.05)elevated after one, two, or three immunizations as compared with controlvalues. These results indicate that in addition to CMI responses,antibody responses directed toward the HER2 protein were also induced inAd5 immune mice immunized with Ad5 [E1-, E2b-]-HER2.

Lack of Adverse Liver Effects in Immunized Mice

Toxicity studies were performed on serum from Ad5 immune mice immunizedwith Ad5 [E1-, E2b-]-HER2 as described above. Ad5 immune mice injectedwith buffer alone served as controls. Three days after the thirdimmunization, aspartate aminotransferase (AST) and alanineaminotransferase (ALT) levels were assessed on the blood samples todetermine liver toxicity due to immunizations (FIG. 13A and FIG. 13B).AST and ALT levels were not elevated over controls followingimmunization with the Ad5 vector. These results demonstrate that micecan be safely immunized with Ad5 [E1-, E2b-]-HER2 to induce specifichumoral and CMI responses against the HER2 antigen.

Challenge of Ad5 [E1-, E2b-]-HER2 Immunized Ad5 Immune Mice with HER2Expressing Tumors

Studies were performed to determine if Ad5 immune mice could first beimmunized with the Ad5 [E1-, E2b-]-HER2 vector platform and resist achallenge of implanted HER2 expressing tumor cells. BALB/c mice wereutilized for these studies. A HER2 expressing BALB/c murine tumor cellline (CT26-HER2) was used. This particular murine cell line is acarcinoma that has been genetically modified to express human HER2 andcan be implanted into BALB/c mice.

Groups of 7 mice each were first injected two times at two weekintervals with Ad5-null to render the mice Ad5 immune. Two weeks afterthe last Ad5-null injection, the mice were immunized three times atweekly intervals with injection buffer (Tumor Controls), Ad5-null(Vector Controls), or Ad5 [E1-, E2b-]-HER2 (Immunized Group). Two weeksfollowing the last immunization (or injection), the mice were implantedsubcutaneously with 1×10⁶ CT26-HER2 cells. After implantation, tumorgrowth was monitored. The tumors were measured and the tumor volumescalculated. As shown in FIG. 14, mice immunized with Ad5 [E1-,E2b-]-HER2 resisted a challenge with HER2 expressing tumor. This was incomparison with receiving injections of buffer only or Ad5-null wherethe tumors grew to a point at which the mice were humanelyexsanguinated. These results demonstrate that Ad5 immune mice immunizedwith Ad5 [E1-, E2b-]-HER2 can significantly slow the progress of tumorgrowth after a lethal challenge of HER2 expressing tumor cells.

Example 6 THE Ad5 [E1-]-CEA Vector Vaccine Induces CEA Specific ImmuneResponse Upon Re-Immunization in Ad5 Immune Mice

This example shows that the Ad5 [E1-, E2b-] vector platform induces CMIresponses against the tumor associated antigen (TAA) carcinoembryonicantigen (CEA) in the presence of pre-existing Ad5 immunity in mice.

Characterization of Ad5 CEA vectors

Initial studies were performed to confirm CEA gene expression of twoAd5-CEA vector platforms. It was first determined that the CEA antigencould be expressed on cells transfected with the vaccine vectorplatforms. A549 cells were obtained from ATCC and transfected with Ad5[E1-]-CEA or Ad5 [E1-, E2b-]-CEA. Western blot analysis revealed thatcells transfected with the vector platforms expressed CEA antigen.

Induction of Ad5 Immunity in Mice

To assess the levels of Ad5 immunity that could be induced, groups ofAd5 naïve C57BL/6 mice were injected subcutaneously with the Ad5 vectorplatform (VP). Twenty eight to forty two days later, serum samples werecollected and assessed for endpoint Ad5 NAb titers. As shown in FIG. 15,undetectable Ad5 NAb titers (endpoint Ad5 NAb titer <1/25) were observedin normal control mice. Ad5 NAb (endpoint titers of 1/25 to 1/50) wasdetectable after one injection but dramatically increased after threeinjections of 10¹⁰ Ad5. Therefore, in additional Ad5 immune studies,mice were injected twice with 10¹⁰ Ad5 VP to render the animals Ad5immune.

Immunization of Ad5 Immune Mice with Ad5 [E1-]-CEA or Ad5 [E1-,E2b-]-CEA.

These experiments were designed to determine and compare theimmunization induction potential of Ad5 [E1-]-CEA and Ad5 [E1-,E2b-]-CEA vaccines in Ad5 immune mice. Groups of female C57BL/6 mice, 4to 8 weeks old, were immunized 2 times at 2 week intervals with 10¹⁰Ad5-null VP. Two weeks following the last Ad5-null immunization, themice were immunized 3 times at weekly intervals with 10¹⁰ VP of Ad5[E1-]-CEA or Ad5 [E1-, E2b-]-CEA. Two weeks following the lastimmunization, mice were euthanized and their spleens and sera harvestedfor analyses.

CMI responses were assessed by ELISpot assays performed on splenocytesexposed to intact CEA antigen. Splenocytes from Ad5 immune C57BL/6 micethat were immunized subcutaneously with Ad5 [E1-]-CEA or Ad5 [E1-,E2b-]-CEA were harvested and assessed for the number of IFN-γ and IL-2secreting cells as described above. As shown in FIG. 16A and FIG. 16B,significantly elevated numbers of both IFN-γ and IL-2 secreting cellswere observed in spleens assayed from mice immunized with Ad5 [E1-,E2b-]-CEA as compared to immunized Ad5 [E1-]-CEA mice. Specificitystudies revealed that immunizations with Ad5 CEA vectors inducedspecific CEA associated CMI responses and not responses against otherirrelevant antigens such as the HIV-gag protein or β-galactosidase.These results demonstrate that immunization of Ad5 immune mice with Ad5[E1-, E2b-]-CEA induce significantly higher CMI responses.

Lack of Adverse Liver Effects in Immunized Mice

Toxicity studies were performed on serum from Ad5 immune female C57BL/6mice immunized with Ad5 [E1-]-CEA, Ad5 [E1-, E2b-]-CEA as describedabove. Ad5 naïve or Ad5 immune mice injected with buffer alone served ascontrols. Three days after the third immunization, aspartateaminotransferase (AST) levels were assessed on the blood samples todetermine liver toxicity due to the treatment. AST levels were notelevated over controls following immunization with either vector (FIG.17). Alanine aminotransferase (ALT) levels were also assessed andsimilar results were observed.

Ad5 [E1-, E2b-]-CEA Immunotherapy in Ad5 Immune Tumor Bearing Mice

Based upon the successful immunological results observed above, studiesin which MC38 tumors were established in mice and then treated wereperformed as described below. For these studies a CEA expressing MC38murine cell line was used. This cell line has been genetically modifiedto express human CEA and can be implanted into C57BL/6 mice. After tumorestablishment, the mice were treated with the novel Ad5 [E1-, E2b-]-CEAvector platform. To determine if Ad5 immune tumor bearing mice could betreated with the Ad5 [E1-, E2b-]-CEA vector, C57BL/6 mice were injectedtwo times subcutaneously with 10¹⁰ Ad5 [E1-]-null VP at 14 day intervalsto render the mice Ad5 immune. Two weeks after the last injection, twogroups of 7 C57BL/6 mice were injected subcutaneously with 10⁶ CEAexpressing MC38 tumor cells. Seven days later, when tumors werepalpable, one group of mice was treated by distal subcutaneous injectionwith 10¹⁰ VP of Ad5 [E1-, E2b-]-CEA on days 7, 13 and 19. A group of 7injection buffer only treated C57BL/6 mice served as untreated controls.All mice were monitored for tumor size over a 21 day period and tumorvolumes were determined as previously described.

As shown in FIG. 18, the tumor growth by day 19 was significantlyreduced in the Ad5 [E1-, E2b-]-CEA treated mice and remained so. At theend of the study (Day 22), the mice were sacrificed and the tumors wereexcised and weighed. As shown in FIG. 19, the tumors in the mice treatedwith Ad5 [E1-, E2b-]-CEA were significantly (P<0.05) smaller in weightthan the untreated controls.

At the termination of the study, spleens were collected from mice andthe CEA specific CMI response was determined by ELISpot assay. CEAspecific IFN-γ secretion response was significantly higher in miceimmunized with Ad5 [E1-, E2b-]-CEA than in mice who received MC-38 tumorcells alone. These results indicate that treatment of CEA expressingtumors in Ad5 immunized mice using the Ad5 [E1-, E2b-]-CEA vaccine cansignificantly decrease tumor growth progression.

Example 7 Immunizations with an Ad5 [E1-, E2B-] Vector Vaccine inNon-Human Primates with Pre-Existing Ad5 Immunity

This example shows that multiple immunizations with an Ad5 [E1-, E2b-]vector vaccine in non-human primates (NHP) with pre-existing Ad5immunity is effective.

An Ad5 [E1-, E2b-]-Simian immunodeficiency virus (SIV)-gag vector wasconstructed by recombination in BJ5183 bacterial cells and rescued bytransfection into pre-terminal protein (pTP) and polymerase (pol)expressing E.C7 cells. The Ad5 [E1-, E2b-] vector contains deletions inboth the pol and pTP genes, since this arrangement has been demonstratedto result in decreased liver toxicity when compared to Ad5 [E1-] virus.The mini-gene cassette expressing the particular antigen is subclonedinto the E1 region of a shuttle vector and recombined with E2b deleted(pol-, pTP-) Ad5 genomic DNA. The SIV gag gene was constructed fromSimian (macaque) immunodeficiency virus, isolate 239 genome (GenBankAccession #M33262.1). The SIV gag (951-1074) containing plasmid wassynthesized by GeneArt (Regensburg, Germany). The recombined Ad5 [E1-,E2b-] vector was verified by restriction analysis and rescued as virusparticles by restriction enzyme release and transfection into E.C7cells. The Ad5 [E1-, E2b-] viral vector was manufactured by releasingfrom E.C7 producer cells by Triton X-100, precipitated in PEG, purifiedon CsCl gradients, dialyzed against 20 mM HEPES (pH 7.4) containing 5%sucrose, aliquoted, and frozen in a dry ice-ethanol bath. Particleconcentration is calculated both by absorption at 260 and 280 nm (normalratio=1.3) and by real-time PCR analysis. The former value is used todetermine amounts of virus for in vivo experiments. The Ad5 [E1-, E2b-]vectors are further tested for antigen expression in infected cells.This is accomplished by transfection of A549 human lung carcinoma cellsin vitro and subsequent analysis of isolated protein by Western blotassay for the detection of expressed antigen in transfected cells.

A total of 18 animals were used in the study. Seven NHP were immunizedtwo times at two weeks intervals with 1×10¹⁰ viral particles (VP) ofAd5-null (empty) to render the animals Ad5 immune prior to immunizationwith the vaccine (Group 1). Seven NHP were not immunized with Ad5-nullto serve as naïve Ad5 [E1-, E2b-]-SIV-gag immunized controls (Group 2).Four NHP were immunized two times at two weeks intervals with 1×10¹⁰viral particles (VP) of Ad5-null (empty) to render the animals Ad5immune and served as Ad5 immune non-Ad5 [E1-, E2b-]-SIV-gag immunizedcontrols (Group 3). Prior to initiation of immunizations, the NHP weretested for the presence of Ad NAb. As shown in Table 1, all NHPpre-immunized with Ad5-null exhibited Ad5 neutralizing activity (NAb)with endpoint titers of 1/100 to 1/200. NHP that were not immunized withAd5-Null had NAb endpoint titers 1/10 or less.

TABLE 1 NHP Endpoint NAb Group 1-NHP-1 1/200 Group 1-NHP-2 1/200 Group1-NHP-3 1/200 Group 1-NHP-4 1/200 Group 1-NHP-5 1/200 Group 1-NHP-61/100 Group 1-NHP-7 1/200 Group 2-NHP-1 1/10  Group 2-NHP-2 <1/10   Group 2-NHP-3 <1/10    Group 2-NHP-4 <1/10    Group 2-NHP-5 <1/10   Group 2-NHP-6 <1/10    Group 2-NHP-7 <1/10    Group 3-NHP-1 1/200 Group3-NHP-2 1/200 Group 3-NHP-3 1/200 Group 3-NHP-4 1/100

All the NHP in Groups 1 and 2 were then immunized subcutaneously twotimes at two week intervals with 1×10¹⁰ VP Ad5 [E1-, E2b-]-SIV-gag. Twoweeks after the second immunization with vaccine, PBMCs were isolatedfrom each animal and numbers of IFN-γ secreting lymphocytes wereassessed by ELISpot assay. As shown in FIG. 20, significantly (P<0.05)elevated numbers of IFN-γ secreting lymphocytes were detected inimmunized NHP as compared with non-immunized Ad5 immune control NHP(Group 3). Moreover, the levels of IFN-γ secreting lymphocytes observedimmunized Ad5 immune NHP were not significantly different from thelevels of IFN-γ lymphocytes observed in non-Ad5 immune NHP. Theseresults indicate that high levels of CMI responses can be achieved inNHP despite the presence of pre-existing immunity to Ad5 and theselevels are comparable to those observed in naïve animals.

Example 8 A Modified E6 Cancer Vaccine Vector Based on the Ad5 [E1-,E2B-] Platform

This example illustrates how multiple immunizations of Ad5 immune micewith Ad5 [E1-, E2b-]-E6E7 could induce HPV E6E7 specific CMI andantibody responses that have beneficial effects on tumorigenesis.

Production and Characterization of Ad5 [E1-, E2b-]-E6E7 Vector

The early gene 6 (E6) of the human papilloma virus (HPV) encodes anoncoprotein involved in the tumorigenesis of HPV. The E6 proteincontributes to tumorigenesis by binding to p53 in human cells. Thisleads to a downregulation of p53 target genes that, in turn, leads tofaulty DNA replication and accumulation of mutations.

The early gene 7 (E7) of HPV encodes an oncoprotein involved in thetumorigenesis of HPV. The E7 protein contributes to tumorigenesis bybinding to retinoblastoma protein (pRb) in human cells. This ultimatelyleads to a disruption in the cell cycle causing uncontrolled cellularproliferation.

The HPV E6 and E7 polynucleotide sequences utilized for these studiesare modified so that the encoded polypeptide remains antigenic but is nolonger oncogenic. For example, the modified E6 peptide may no longer beable to interact with p53 (e.g., deletion of the p53 binding site) butis capable of generating an immune response against the unmodified E6protein. For transgene construction in these studies, the p53 bindingsite of the constructed gene will be eliminated. The p53 binding sitemay be eliminated by mutating or deleting a portion of the E6 protein.Similarly, the modified E7 peptide will no longer be able to interactwith pRb (e.g., deletion of the pRb binding site), but will be capableof generating an immune response against the unmodified E7 protein.Therefore, the pRb binding site will be eliminated for transgeneconstruction. The pRb binding site may be eliminated by mutating ordeleting a portion of the E7 protein.

The modified E6 and E7 constructs could each be used separately forvaccines; however, the study described herein uses a construct encodingboth E6 and E7, to be expressed either as separate polypeptides or as afusion polypeptide.

Initial studies are performed to confirm gene expression of the Ad5[E1-, E2b-]-E6E7 vector platform. To determine that E6E7 is expressed bycells transfected with the vaccine vector platform, A549 cells availablefrom ATCC are transfected with Ad5 [E1-, E2b-]-E6E7. Western blotanalysis is used to determine if cells transfected with the vectorplatform express E6E7. Briefly, 10⁶ human lung carcinoma cells (A-549)(ATCC number CCL-185) are infected at a multiplicity of infection (MOI)of 900 or 150 VP, incubated for 24 hours and then lysed. Cell lysatesare separated on a 10% SDS-polyacrylamide gel and transferred onto aPVDF membrane (GE Heathcare, Piscataway, N.J.). The membranes are thenblocked with TBS containing 5% (w/v) blocking reagent (GE Healthcare,Piscataway, N.J.) for 2 hours at room temperature and sequentiallyincubated with mouse anti-HER2 antibody (1:250) (Genway, San Diego,Calif.) and goat anti-mouse-HRP conjugated antibody (1:1000) (Kirkegaard& Perry Laboratories, Gaithersburg, Md.) for one hour at roomtemperature. Reactivity is determined by chemiluminescence using an ECLWestern Blotting analysis system (GE Healthcare, Piscataway, N.J.)according to the manufacturer's specifications. It is expected that E6E7protein bands will be observed after development.

Escalating Dose Trial of Ad5 [E1-, E2b-]-E6E7 Vaccine:

A study is performed to determine the effect of immunizations withvarious doses of Ad5 [E1-, E2b-]-E6E7 on the induction of CMI responsesin mice. Groups of naïve female BALB/c mice, 4 to 8 weeks old, areimmunized three times at weekly intervals with 10⁸, 10⁹, or 10¹⁰ VP ofAd5 [E1-, E2b-]-E6E7. Control mice are injected with buffer solutiononly. Two weeks after the last immunization, the mice are euthanized andCMI responses assessed. A dose response effect is expected to beobserved with the highest CMI response levels observed afterimmunizations with 10¹⁰ viral particles (VP) of Ad5 [E1-, E2b-]-E6E7.

Induction of Ad5 Immunity in Mice as Evidenced by Levels of Ad5Neutralizing Antibody

To induce Ad5 immunity, Ad5 naïve mice are injected intradermally twiceat two week intervals with 10¹⁰ virus particles of Ad5-null (empty). Twoweeks later, serum samples are collected and assessed for endpoint Ad5neutralizing antibody (NAb) titers. As described above and shown in FIG.9, this protocol induces NAb activity in mice. Therefore, mice will bemade Ad5 immune by two injections with Ad5-null.

Immunization of Ad5 Immune Mice with Ad5 [E1-, E2b-]-E6E7

Experiments are performed to assess immune responses in Ad5 immune miceusing multiple immunizations with Ad5 vector based vaccines. Two weeksfollowing the last Ad5-null (empty vector) immunization, groups offemale BALB/c mice, 4 to 8 weeks old, are immunized one, two or threetimes at weekly intervals with 10¹⁰ virus particles of Ad5 [E1-,E2b-]-E6E7. Thus, before mice are immunized with Ad5 [E1-, E2b-]-E6E7,they will have measurable pre-existing Ad5 NAb endpoint titers. Twoweeks following the last immunization with Ad5 [E1-, E2b-]-E6E7, miceare euthanized and their spleens harvested for analysis of CMI andantibody responses over the course of one, two, and three immunizationswith Ad5 [E1-, E2b-]-E6E7.

Induction of CMI Responses

CMI responses will be assessed by ELISpot assays performed onsplenocytes exposed to E6 and E7 peptides. Splenocytes from Ad5 immunemice that were immunized with Ad5 [E1-, E2b-]-E6E7 are harvested andassessed for the number of IFN-γ and IL-2 secreting splenocytes. Anelevation in numbers of IFN-γ and IL-2 secreting cells in spleensassayed from mice after immunizations with Ad5 [E1-, E2b-]-E6E7indicates an E6E7-specific CMI response.

Specificity studies can be used to confirm that immunizations inducedspecific E6 and E7 associated CMI responses and not responses againstother irrelevant antigens. These assays can demonstrate that multipleimmunizations of Ad5 immune mice with Ad5 [E1-, E2b-]-E6E7 induces E6and E7 specific CMI responses.

Induction of Humoral Responses

Studies are also performed to determine if anti-E6 and anti-E7 IgGantibodies are induced after immunizations. For example, an ELISA forcirculating IgG antibody to E6 and E7 can be performed. Increasingquantities of detectable antibody to E6 and E7 observed after one, two,or three immunizations with Ad5 [E1-, E2b-]-E6E7 indicate that inaddition to CMI responses, antibody responses directed toward the E6 andE7 proteins are also induced in Ad5 immune mice immunized with Ad5 [E1-,E2b-]-E6E7.

Detection of Adverse Liver Effects in Immunized Mice

Toxicity studies are performed on serum from Ad5 immune mice immunizedwith Ad5 [E1-, E2b-]-E6E7 as described above. Ad5 immune mice injectedwith buffer alone serve as controls. Three days after the thirdimmunization, aspartate aminotransferase (AST) and alanineaminotransferase (ALT) levels are assessed in the blood samples todetermine if any liver toxicity due to the immunizations is present. IfAST and ALT levels are not elevated over controls following immunizationwith the Ad5 vector, it indicates that mice can be safely immunized withAd5 [E1-, E2b-]-E6E7 to induce specific humoral and CMI responsesagainst E6 and E7.

Tumor Challenge of Ad5 [E1-, E2b-]-E6E7 Immunized Ad5 Immune Mice

Studies are performed to determine if Ad5 immune mice immunized with theAd5 [E1-, E2b-]-E6E7 vector platform can resist a challenge of implantedtumor cells expressing E6 and/or E7.

Groups of 7 mice each are injected two times at two week intervals withAd5-null to render the mice Ad5 immune. Two weeks after the lastAd5-null injection, the mice are injected three times at weeklyintervals with injection buffer (Tumor Controls), immunized withAd5-null (No Transgene, vector control group) (Vector Controls), or withAd5 [E1-, E2b-]-E6E7 (Immunized Group). Two weeks following the lastimmunization (or injection), the mice are implanted subcutaneously with1×10⁶ E6E7 expressing murine tumor cells. After implantation, tumorprogression is monitored. The tumors are measured and the volumescalculated. If mice immunized with Ad5 [E1-, E2b-]-E6E7 resist achallenge with E6E7 expressing tumor in comparison with mice receivinginjections of buffer only or Ad5-null, it indicates that Ad5 immune miceimmunized with Ad5 [E1-, E2b-]-E6E7 can slow the progress of tumorgrowth after a lethal challenge of E6E7 expressing tumor cells. It isexpected that tumor progression will occur in the tumor control andAd5-null vector control groups. In contrast, little or no tumor growthor progression is expected to be observed in the Ad5 [E1-, E2b-]-E6E7treated group.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent application, foreign patents, foreign patentapplication and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, application and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

What is claimed is:
 1. A composition comprising a replication defectiveadenovirus vector comprising: a deletion in an E2b region of thereplication defective adenovirus vector; and a nucleic acid sequenceencoding a tumor associated antigen, wherein the tumor associatedantigen is a HPV E6 antigen, a HPV E7 antigen, or a combination thereof.2. The composition of claim 1, wherein the replication defectiveadenovirus vector further comprises a deletion in an E1 region, adeletion in an E3 region, a deletion in an E4 region, or a combinationthereof.
 3. The composition of claim 1, wherein the replicationdefective adenovirus vector is not a gutted vector.
 4. The compositionof claim 1, wherein the composition comprises the replication defectiveadenovirus vector at a concentration of at least 10⁶ virus particles/ml.5. The composition of claim 1, wherein the composition comprises thereplication defective adenovirus vector at a concentration of at least10⁷ virus particles/ml.
 6. The composition of claim 1, wherein thecomposition comprises the replication defective adenovirus vector at aconcentration of at least 10⁸ virus particles/ml.
 7. The composition ofclaim 1, wherein the composition comprises the replication defectiveadenovirus vector at a concentration of at least 10⁹ virus particles/ml.8. The composition of claim 1, wherein the composition comprises thereplication defective adenovirus vector at a concentration of at least10¹⁰ virus particles/ml.
 9. The composition of claim 1, wherein thecomposition comprises the replication defective adenovirus vector at aconcentration of at least 10¹¹ virus particles/ml.
 10. The compositionof claim 1, further comprising an immune adjuvant.
 11. The compositionof claim 1, wherein the composition is formulated in a liposome.
 12. Thecomposition of claim 1, wherein the HPV E6 antigen or HPV E7 antigen isnon-oncogenic.
 13. The composition of claim 1, wherein the HPV E6antigen has a deletion of its p53 binding site.
 14. The composition ofclaim 1, wherein the replication defective adenovirus vector generatesan immune response to the tumor associated antigen following multiplerounds of immunization with the same type of replication defectiveadenovirus vector.
 15. A replication defective adenovirus comprising agenome deletion in an E2b region; and a nucleic acid coding sequence,wherein the improvement comprises: the nucleic acid coding sequenceencodes an HPV E6 antigen, such that the adenovirus is capable ofgenerating an immune response to HPV E6 antigen following administrationof the adenovirus to an individual having preexisting immunity toadenovirus.
 16. A replication defective adenovirus comprising a genomedeletion in an E2b region; and a nucleic acid coding sequence, whereinthe improvement comprises: the nucleic acid coding sequence encodes anHPV E7 antigen, such that the adenovirus is capable of generating animmune response to HPV E7 antigen following administration of theadenovirus to an individual having preexisting immunity to adenovirus.